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I. INTRODUCTION
Proteins are biomacromolecules that play essential roles in life processes spanning from
metabolic process regulation, cellular information exchange, cell-cycle control, and molecular
transport to protection from the environment.1 In biomedicine, for example, proteins are of
great interest as disease biomarkers. In biotechnology, as another example, the role of enzymes
as biocatalysts is a topic of much study. Owing to functional involvement in physiological proc-
esses, protein state (expression levels and modifications) may be effective indicators of a dis-
ease state and/or response to therapeutic treatment.2 Biomarker detection using immunoassays
has been a widely used disease diagnostic tool.3 Promising protein biomarkers benefit from fur-
ther characterization by immunoassays and similar analytical tools.4 Immunoassays exploiting
specific recognition of protein biomarkers by cognate antibodies have been optimized for high
analytical performance (e.g., rapid assays, label-free detection, improved limits of detection,
and multiplexing capability). Enzymes are a specific class of proteins that catalyze biochemical
reactions. Enzymes display selectivity, accelerate reactions, provide environmentally friendly
means to organic synthesis, and effectively synthesize complicated biomolecules such as DNA
a)
Author to whom correspondence should be addressed. Email: aeh@berkeley.edu
and RNA.5 As enzymes are selective and effective proteinaceous biocatalysts that convert sub-
strates into products, the enzyme is actively used across agricultural feeds, polymer synthesis,
biofuels production, food processing, and the paper industry.6 Enzymes are also used widely in
biosciences and biotechnology such as genetic engineering (e.g., oligonucleotide manipulation)
and the pharmaceutical industry (e.g., production of pharmaceutical ingredients).6,7 In addition,
enzyme-mediated fluorescence or colorimetric detection of proteins, i.e., ELISA (Enzyme-
Linked Immunosorbent Assay), is a standard immunoassay technique.
In analysis of proteins and enzymes, microfluidic design has proven to be a powerful tech-
nological tool to improve performance of immunoassays,810 enzymatic reactors,1113 and other
biological assays.14 Importantly, manipulation of liquid inside microscale fluidic networks ena-
bles reduced consumption of reagents, compared to macroscale instruments.810,13,14 Decreased
liquid volume and short diffusion lengths allow facile reactions between analyte and antibody
or enzyme and substrate, resulting in reduced assay times.8,9,11 Using design strategies pio-
neered by the semiconductor industry, microfluidic integration offers a sample-in, answer-out
capability.9,10,12,1417 Microfluidic technologies make possible monolithic integration of disjoint
assay steps, further underpinning automation of those steps.1524 As discussed in depth in this
review, the fine spatial control in immobilizing proteins and biomolecules inside microchannels
allows multiplexed21,22,25 and multiparameter assays.26 The overall form factor of self-
contained microfluidic devices (and automation) reduces human errors and risks of exposure to
dangerous and toxic bio-/chemical reagents.
Analytical immunoassays in microfluidic formats are designed for rapid and sensitive
detection of one or several targeted antigens in clinical diagnostics,2733 as protein sensors,3437
or in environmental analysis.3842 Laboratory-grade assays such as polyacrylamide gel electro-
phoresis (PAGE) based immunoassays,4345 isoelectric focusing (IEF),21 and Western
blotting1820,22,24,46 provide qualitative and/or quantitative information on multiple proteins,
even in complex biological fluids. Questions spanning from protein-protein interactions,47,48
and protein binding kinetics,49,50 to post-translational modifications23,51 have all been pursued
using analytical technologies in microfluidic formats. Recent reviews by Hanares et al.,9 Bange
et al.,10 and Ng et al.8 are recommended as excellent overviews of immunoassay advances.
Microfluidic enzyme reactors find use in analysis and optimization of biocatalytic process. For
more detailed information on microfluidic enzyme reactors, recent reviews by Krenkova
et al.,11 Asanomi et al.,12 and Miyazaki et al.13 are recommended. Here, before scaling up to a
large-scale batch process, the throughput and appreciable assay sensitivity of a microfluidic for-
mat can expedite candidate-enzyme screening process from mutant libraries.52 Enzyme-kinetic
study has been performed in microfluidic formats.5358 Important to proteomics, enzymatic
digestion before MALDI-TOF/MS (Matrix-assisted laser desorption-ionization time-of-fly/mass
spectrometry) peptide mapping of a protein has been explored in microfluidic devices.5966
Compared to conventional in-solution enzyme digestion, which is time consuming and offers
limited sensitivity, microfluidic formats have shown high conversion rates, facile replacement
of inactivated enzyme, and long-term stability.54,55,63,67 Enzymatic production of fluorescent
and colored products for protein analysis (e.g., alkaline phosphatase (ALP) production of chem-
iluminescence or colorimetric product) enhances detection limits of immunoassays.
Heterogeneous assay formats where one binding or reaction partner is immobilized to a sur-
face are widely employed and are the focus of this review. Consequently, surface immobilization
is a primary design and performance consideration.813 In contrast to heterogeneous formats are
homogeneous approaches, where reactants are reacted in solution. This review focuses on
the former category of reactions. Two examples of heterogeneous assays that are popular in
microfluidic formats are immunoassays810 and enzyme reactors.1113 For microfluidic immuno-
assays, antigen or antibody is immobilized inside microchannels. Key immunoassay performance
metrics include analytical sensitivity, analytical specificity, and reproducibility. The immunoas-
say performance depends on the quality of protein immobilization, and thus on the immobiliza-
tion surface, immobilization chemistry, and surface passivation technique (i.e., antibiofouling).68
In microfluidic enzymatic reactors where enzyme is immobilized inside microchannels, the
enzyme conversion rate, long-term stability, and reusability depend on similar immobilization
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factors.63 We will cover the design and operation of these two canonical heterogeneous for-
matsthe immunoassay and enzyme reactorsas we detail design and operational considera-
tions for protein immobilization in microfluidic systems.
FIG. 1. Role of surface geometry in binding site density. Schematic drawing of (a) planar and (b) high surface-area-to-vol-
ume ratio three-dimensional immobilization surfaces.
041501-4 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
owing to low cost, simple assay visualization, and simple reagent immobilization.103105 In this sec-
tion, we provide more detail on the most popular immobilization surfaces.
1. Silicon
Silicon is a most widely used and well-characterized substrate originating from integrated
circuit development in the semiconductor industry. Silicon was adapted as a mechanical mate-
rial with the advent of microelectromechanical systems (MEMS)108 and, subsequently,
employed in the first microfluidic analytical system.109 Owing to high-resolution microfabrica-
tion technique development (feature size as low as 22 nm are attainable with mass fabrica-
tion110), fine fluidic channels and microfluidic components can be created on a silicon substrate.
Naturally or artificially grown oxide on the silicon surface makes silanol-based chemistries
compatible with for protein immobilization on silicon.40,111,112 While powerful, silicon has,
however, three major drawbacks for microfluidic design: (1) opaqueness of silicon in the visible
spectrum renders various optical imaging techniques irrelevant; (2) incompatibility of silicon
with electrokinetic methods owing to the electrical conductivity of the silicon substrate, and (3)
expense associated with the sophisticated microfabrication techniques used to micromachine sil-
icon in a cleanroom environment. Therefore, silicon is not as widely used as initially with the
exceptions of continued widespread application in electrochemical analysis113 and SPR.65
2. Glass
Besides silicon, glass (e.g., fused silica, soda lime, borosilicate glass), and quartz are
another widely used substrate for microfluidic devices.114 Glass substrates were initially used in
microfluidic electrophoresis systems, building on the earlier successes of glass capillaries as a
format of choice among the electrophoresis community.115 Glass is transparent across a wide
spectrum, with negligible autofluorescence.74 Therefore, glass is well-suited for fluorescence-
based microfluidic assay readouts. Glass is robust, being resistant to solvents and acids and
bases.10 Several commercially successful microfluidic devices use glass substrates.116 However,
glass is not without disadvantages. Glass can fracture, so it must be handled with a care. Glass
microfabrication can be costly, since much of the process requires a cleanroom facility.
Microfabrication processes similar to those used for silicon result in patterned microchannels
but acid wet etching of glass does not yield high-aspect-ratio anisotropic microchannels unlike
silicon (e.g., deep reactive-ion etching process). Glass substrates benefit from immobilization
chemistries including various silanol chemistries for covalent linking of proteins.117
3. PDMS
PDMS is currently one of the most frequently used and studied substrates in microfluidics,
owing to a rapid design-fabricate-test cycle and low cost. PDMS is a rubber-like flexible
041501-5 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
polymer (i.e., elastomer) and is transparent making optical imaging possible. In contrast to rigid
substrates such as silicon and glass, microfluidic actuators such as valves and pumps can be
readily formed in a microdevice.118 Owing to rapid casting-based soft lithography processing,
fabrication of microfluidic networks in PDMS is inexpensive, requiring low investment in infra-
structure. Overall, PDMS is an excellent material for rapid prototyping of research device.
After treating with oxygen plasma, PDMS can be irreversibly sealed with glass, plastic sub-
strates, or PDMS slabs to form enclosed microchannels. Multiple layers of PDMS can be
stacked yielding multifunctional microfluidic devices.118 This fabrication process stands in stark
contrast to complex and time-consuming silicon or glass bonding processes.89,93 Nevertheless,
no material is well suited to every application. Drawbacks of PDMS are as follows: limited re-
sistance to organic solvents, gas permeability, compliant characteristics and thus often inappro-
priate for harsh environments needing robust packaging.10 As related to protein immobilization,
PDMS is hydrophobic in native form, so proteins tend to readily and nonspecifically bind to the
surface. Therefore, blocking of the adsorptive surface must be done before an assay is com-
pleted. PDMS lacks functional groups for covalent derivatization. Silanol groups can be intro-
duced after oxygen plasma treatment but these groups do not offer long-term stability.119
Therefore, immobilization methods are generally complex and require multiple steps to imple-
ment.97,120 Because a large numbers of microfluidic devices are made by sealing microchannel-
patterned PDMS slabs to glass slides, consideration of glass and PDMS surface properties is
often required (e.g., care to avoid nonspecific adsorption to PDMS surfaces when protein is im-
mobilized on glass surface).
4. Plastic
With a relevance to mass fabrication, cost effective, robust, and reliable substrates for
microfluidics are of great interest. Microfluidic chips fabricated from plastics such as PMMA,
PS, and COC are extremely cheap to mass produce when using mold-based techniques such as
injection molding or hot embossing.76 Moreover, plastic is generally resistant to solvents and
acids/bases, rigid but not fragile, common in the marketplace, and optically transparent.7476
Owing to these attributes, numerous groups have investigated plastic as a material of a choice
for commercial microfluidic devices.121,122 Indeed, a few commercial microfluidic devices are
made of plastic.123 Plastics share the disadvantages of PDMS, being hydrophobic in native
form making hydrophobic nonspecific protein adsorption a concern. Inert plastic surfaces lack
functional groups, so chemistry is employed to prepare the surface to immobilize pro-
teins.29,80,124 Oxygen plasma77,79 or strong bases/oxidizers35,77,78 are often used to introduce
functional groups.
5. Metal
Metals films are sometimes deposited on silicon or glass surface. Protein is immobilized on
the metal surface for detection methods other than fluorescence or colorimetric detection.
Several assay readout modes are appropriate, including electrochemical sensing,35,36,69 Raman
spectroscopy,69 and SPR.8,9,49 Thiol-based chemistry, although not as strong as covalent link-
ages, is available for protein immobilization on noble metal surfaces including gold, silver, and
platinum.125
2. Hydrogel
Hydrogels have been actively used in tissue engineering129 and protein microarray81 owing
to the hydrophilic, protein-friendly microenvironment offered. Hydrogels are flexible materials
with a well-ordered fibrous structure. Synthetic PEGDA (PEG diacrylate) gel57,83 and polyacryl-
amide gel1824,9496 are popular with natural gels like chitosan130 or agarose gel also used.
Porogen is sometimes employed to further increase the surface area by inclusion of macro-
pores.82,83 Hydrogel is usually transparent so that sensitive fluorescence imaging is appropriate.
Polyacrylamide gel can also act as a biomolecular sieve without significant nonspecific adsorp-
tion, so protein can be separated based on charge and/or size (e.g., SDS-PAGE or sodium-do-
decyl-sulfate polyacrylamide gel electrophoresis) before a detection step.2224,131,132 With suita-
ble surface modification, gel regions offering different assay functions can be integrated on a
single chip using fabrication via photopatterning.1820,23,24,46,94 The swelling property of hydro-
gels allows integration of actuators such as valves, allowing integration of sophisticated fluid
handling functions.133 A wide range of immobilization methods are available to hydrogels,
including copolymerization of proteins,18,19,23,94,132 activation for covalent linking of pro-
teins,134 or electrostatic capture on charged hydrogel.20,24 Even with a 3-D microchannel-filling
hydrogel, the microchannel surface should be functionalized for covalent anchoring of the
hydrogel structure within the channel, so that the gel will not drift out of the channel under
hydrodynamic pressure or applied electric field.46,82,83 Hydrogels provide a hydrated environ-
ment for proteins so that native activity or functionality can often be maintained.102 A disad-
vantage of hydrogels is the fragility of some gel structures (i.e., application of shear forces or
high electric fields can damage the structure135). Once formed, a stationary hydrogel is difficult
to remove from the channel if regeneration of the assay system is needed.46
041501-7 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
3. Sol-gel
Sol-gels are a condensation polymerization of colloids in aqueous media. After preparation
of the sol (i.e., metal alkoxide monomer in acid and organic/aqueous solvent), the gel is
formed by polymerization during evaporation of the solvent. By simply adding proteins to the
sol before gelation, proteins are encapsulated in optical transparent 3-D nanostructure. Silica
sol-gels made from silicon alkoxide colloids98 are the most common, but titania64 or zeolite99
colloids are also used. Immobilization of proteins proceeds under mild conditions,64 thus pro-
teins can retain near-native activity.
5. Membrane
Membranes are porous (planar) sheets that can provide a large surface area for protein
immobilization. Commercial membrane comprised of polycarbonate (PC), nitrocellulose (NC),
and PVDF (polyvinylidene fluoride) have been inserted and physically clamped between a
microfluidic chip patterned with channels and a blank cover chip.67,84,85 However, resolving
fluid leakage issues from the clamped membrane can be challenging.84 Membranes can also be
formed in situ by condensation100 or electrospinning.33,136 Membranes have been extensively
used in biochemistry or analytical chemistry to adsorb proteins. Such protein immobilization
strategies rely on intermolecular forces137 (i.e., hydrophobic, electrostatic, and van der Waals)
and simple adsorption without complicated chemical activation of the solid supports.
Membranes also have additional useful properties such as ion selectivity and selected transport
of small molecules such as enzyme substrate (i.e., size exclusion)100 and ions.
6. Paper
Paper, a cellulose membrane, offers a versatile, low-cost material for immobilization.103105
Originated from disposable lateral-flow immunoassays, paper microfluidic devices have the
potential to be more cost-effective than plastics or PDMS.138 Fluid and material transport in
and through paper can be accomplished passively (without power consumption) using capillary
action.103,138 Even though paper is macroscopically planar, the material has microscopic 3-D
pores. Immobilization strategies on paper are rather simple. Much like NC membrane, paper
adsorbs proteins via a combination of intermolecular forces making simple reagent spotting
effective for protein immobilization. Paper is optically opaque, so that sensitive detection using
fluorescence imaging could be difficult. Therefore, the limit of detection is rather poor com-
pared to immunoassays based on transparent substrates.138
7. Porous silicon
Porous silicon is produced by anodic electrochemical or photochemical etching of silicon
in hydrofluoric acid (HF).139 Nanopores in porous silicon are usually straight (unidirectional),
and perpendicular to the silicon surface. Porous silicon offers the advantage that that porosity
and pore size are exquisitely controllable. Owing to sensitive refractive index change, porous
silicon offers exceptional performance an optical biosensor.139 Proteins can be immobilized on
041501-8 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
the porous silicon using covalent chemistries similar to those used for planar silicon
substrates.40,60,68
A. Physisorption
The simplest approach to immobilizing a protein on a surface is physisorption (i.e., physi-
cal adsorption). Protein can be conveniently adsorbed to various surfaces via intermolecular
forces such as electrostatic, hydrophobic, van der Waals, hydrogen bonding interactions, or
combination of those (Figure 2(a)).140 Incubation of protein in solution contacting the immobili-
zation surface or continuous flow of solution will achieve attachment of protein without compli-
cated chemistry or reagents. Physisorption is generally weak, and thus an adsorbed layer of
FIG. 2. Common surface immobilization methods for heterogeneous assays. Schematic of immobilization mechanisms: (a)
physisorption, (b) bioaffinity interaction, and (c) covalent bond. The surface immobilization methods are often used in con-
junction with (d) spacer for improved protein activity.
041501-9 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
proteins is not as stable as one formed by covalent or bioaffinity binding. The intermolecular
forces are highly dependent on environmental condition such as pH, ionic strength, temperature,
and surface condition.107 Therefore, immobilization of proteinsin a reproducible manner
can be difficult using physisorption. As protein immobilization is randomly oriented on the sur-
face, some fraction of the binding sites within a population of immobilized proteins are likely
not accessible.106 Further, protein can be immobilized to the surface via multiple binding sites,
which may result in conformational change and reduction of protein activity.106,107
Immobilization density depends on protein size, as well as physicochemical surface properties.
If the immobilization density is too high, active sites could be sterically blocked.106 Therefore,
the use of a spacer (e.g., PEG) with a surface-attaching head group and a protein-binding tail
group has been widely adopted to reduce steric hindrance (Figure 2(d)).40 Blocking of uncoated
surface should be performed after protein immobilization (e.g., BSA or bovine serum albumin)
in order to minimize nonspecific adsorption of off-target biomolecules. Even given the low
degree of control, many studies have employed physisorption of protein to a microfluidic-
device surface. Popularity of the approach stems from several advantages including simple
assay procedure, no toxic reagents and no sophisticated chemical protocols.125 For plastic and
PDMS, proteins are often adsorbed onto the bare surface owing to the hydrophobic nature of
these substrates. In such systems, intermediate molecules are frequently used in order to attach
to surface by covalent linkage on one end and provide hydrophobic or charged functional group
on the other end for protein physisorption. Surfaces are modified to have stronger charge or
hydrophobicity to adsorb protein better than the native surface, with reports of such physisorp-
tion as practically irreversible.92 In some cases, physisorption happens instantaneously (i.e.,
high kon) compared to covalent or bioaffinity bonds that usually requires a substantial incuba-
tion time, and thus physisorption can be used as an intermediate immobilization step of a multi-
step assay sequence (e.g., Western blot).20,24 Physisorption has a wider choice of buffer system,
compared to widely used covalent bonding through primary amines where popular amine-based
buffers (e.g., Tris, glycine) cannot be used.20
1. Electrostatic interaction
Electrostatic or ionic interaction is fundamental to biomolecular attraction (e.g., protein-
protein interaction141 and DNA hybridization142). Thus, electrostatic interactions are exploited
frequently in biochemical assays, for example, cell adherence to positively charged poly-L-ly-
sine (PLL)-treated PS surfaces143 or protein blot to positively charge nylon membrane.144
Typical positively charged functional groups encountered in biochemistry are protonated amine
(NH3) and quaternary ammonium cations (NR4). Negatively charged functional groups are
carboxylic acid (ACOO) and sulfonic acid (ARSO3). These functional groups are involved
in the electrostatic interaction between protein and a surface. Complete isolation of contribu-
tions from other intermolecular forces may be difficult or impossible. However, electrostatic
interactions can be studied by measuring the binding isotherm while changing the buffer ionic
strength.141,145 Electrostatic interactions find application in the protein immobilization in micro-
fluidic assays. Protein friendly, gel-like hydrophilic environment have been created by electro-
static layering of polyelectrolytes such as PEI [poly(ethylene amine)], PDADMAC [poly(dial-
lyldimethylammonium chloride)], PAA [poly(acrylic acid)], PAH [poly(allylamine
hydrochloride)], and PSS [poly(styrene sulfonate)] on protein-unfriendly hydrophobic polymeric
(e.g., PDMS, PMMA, and PS) surfaces35,7779,124 or silicon surfaces.35,40,59,68,77,79,124 Proteins
are immobilized on the composite layer directly by electrostatic interaction,146 covalent chemis-
try,40,68 or bioaffinity interaction.35,40,68,77,79,124 Electrostatic interaction has also been used to
pack microbeads (with bead-surface immobilized proteins) into beds in microfluidic channels.92
Protein has also been directly immobilized inside hydrogels via electrostatic interaction. Kim
et al. created negatively charge polyacrylamide (PA) gels to immobilize proteins after separa-
tion by electrophoresis to yield microfluidic Western blotting (Figure 3).20 Because of a high
surface charge (120 e) in electrophoresis buffer (pH 8.3), the enzyme b-gal (b-galactosidase)
was copolymerized in PA gel to introduce a negative charge to the gel. CTAB
041501-10 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 3. Charged PA gel allows electrostatic immobilization of CTAB-coated proteins in a microfluidic Western blotting.
Negatively charged PA gel immobilizes separated proteins, followed by fluorescent detection (immunoblotting). Reprinted
with permission from D. Kim et al., Anal. Chem. 84, 2533 (2012). Copyright 2012 American Chemical Society.
2. Hydrophobic interaction
As some designers of novel microfluidic devices aim for disposable point-of-care diagnos-
tics applications, immobilization of biomolecules on polymeric hydrophobic materials has
gained attention. COC, PMMA, and PStransparent, hydrophobic thermoplasticshave gained
attention recently for the POC application.121,122 Bhattacharyya and Klapperich introduced a
hot-embossed COC microfluidic chip for an immunoassay of CRP (C-reactive protein).28
Human CRP was introduced in the microfluidic channel and physisorbed by hydrophobic inter-
action, followed by BSA blocking, and chemiluminescence detection by horse radish peroxidase
(HRP) conjugated antibody.
Tsougeni et al. recently presented an approach to increase protein adsorption on hydropho-
bic PMMA surfaces.149 Using directional O2 plasma etch and mask-based lithography, the
researchers not only patterned microchannels but also roughened the channel surface. The
roughened PMMA surface yielded stronger adsorption of protein (biotinylated BSA or IgG)
compared to a smooth, hot-embossed PMMA surface (i.e., 120 poorer detection limit). Sia
et al. reported a microfluidic device consisting of a PS lid mated to a PDMS substrate patterned
with microchannels.150 HIV Env antigen (gp41) was adsorbed to the PS surface to assay anti-
bodies in HIV-infected sera, with catalytic silver deposition using gold nanoparticle conjugated
secondary antibodies. Xiang et al. designed an H-channel glass-covered PDMS chip.34 An
Escherichia coli antigen was physisorbed to a PDMS surface and then later detected by primary
and secondary antibodies. The authors used electrokinetic fluidic control.
Kitamoris group published work on proteins absorbed to PS microbeads in a bead-based im-
munoassay.89,93 Three glass substrates were patterned with channels using a CO2 laser and fast
atom beam. Then, all the layers were thermally bonded to form a glass microfluidic device.
041501-11 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
Using a dam structure, analyte-adsorbed beads were pseudo-immobilized. Then, gold nanoparticle
functionalized detection antibody was introduced for TLM (thermal lens microscopy). In a first
study, secretory IgA was immobilized then analyzed by detection antibody.93 In a subsequent
study, anti IFN-c (interferon-r) capture antibody was immobilized, and IFN-c was captured. Then
biotinylated detection antibody was introduced, followed by injection of streptavidin conjugated
with gold nanoparticle for improved detection limit.89 Fengs group published on a novel protein-
aceous monolayer for antibody immobilization on a PDMS surface (Figure 4).151,152 The authors
used a copper TEM grid (i.e., stencil) to pattern hydrophobin, allowing conversion of the hydro-
phobic surface to a hydrophilic surface. Hydrophobin is a cysteine-rich small protein (100
amino acids) extracted from filamentous fungi. Hydrophobins can form self-assembled monolayer
on hydrophilic-hydrophobic interface (e.g., air-water interface) owing to amphiphilic nature.
Hydrophobic patches faced towards the PDMS while hydrophilic patches faced away. Chicken
IgG was physisorbed via polar interaction to the hydrophobin SAM (self-assembled monolayer)
creating a heterogeneous immunoassay format. Jo et al. reported a mass spectrometric (MS)
imaging (spatially resolved MS information of attached polypeptides) modality using a PDMS
microfluidic device.65 An Aplysia bag cell was attached to a PLL coated silicon surface through
electrostatic interaction. Released neuropeptides were delivered through a PDMS microfluidic
channel and physisorbed to another silicon surface rendered hydrophobic using octadecyltrichloro-
silane (OTS) treatment. The peptides bradykinin, angiotensin II, substance P, renin substrate, and
egg laying hormone were imaged by MALDI-TOF/MS.
As described earlier, the simplicity of physisorption makes it a preferred method for immo-
bilizing proteins in early, proof-of-concept experimentsa prototyping immobilization strategy.
The Whitesides group created a 3-D microfluidic stamp in PDMS as the basis for their tech-
nique called 3-D micromolding in capillaries (MIMIC), which overcame limitations of con-
ventional soft lithography.153 The micro contact printing can pattern complex protein patterns
but requires multiple inking and stamping steps to have discrete pattern of multiple protein spe-
cies.154 2-D MIMIC technique can deposit a discrete pattern of multiple protein species at a sin-
gle stamping, but the pattern has to be continuous because the technique uses microfluidic
channels that does not cross over.155 On the contrary, the 3-D MIMIC can put a discrete pattern
of multiple proteins in a single stamping.153 BSA and fibrinogen were physisorbed to a PS sub-
strate, and a complex protein pattern was created (Figure 5(a)). Following up on early work on
microfluidic networks (lFN),156 Delamarche created a simple protein-microarray-like multi-
plexed immunoassay.157 The immunoassay was enabled by reversible sealing of silicon lFN to
a PDMS slab, with simple physisorption of proteins resulting in a striped pattern on the PDMS.
After patterning, the cover (housing a series of trenches) was rotated 90 and another reversible
sealing of the lFN on the PDMS slab created enclosed microchannels for introduction of detec-
tion antibody and a multiplexed immunoassay was completed (Figure 5(b)). In later work,
FIG. 4. Immobilization process of chicken IgG on hydrophobin coated PDMS surface and immunoassay. Reprinted with
permission from R. Wang et al., Chem. Mater. 19, 3227 (2007). Copyright 2007 American Chemical Society.
041501-12 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 5. Novel protein patterning methods using simple physisorption: (a) nested spirals of BSA (bright green) and fibrino-
gen (light green) on a PS surface using 3-D MIMIC technique. Reprinted with permission from D. T. Chiu et al., Proc.
Natl. Acad. Sci. U.S.A. 97, 2408 (2000). Copyright 2000 National Academy of Science of USA. (b) Multiplexed immuno-
assay using lFN and reversible PDMS-to-PDMS sealing. Reprinted with permission from A. Bernard et al., Anal. Chem.
73, 8 (2001). Copyright 2001 American Chemical Society. (c) Protocol for an immunoassay in which the protein capture
sites are patterned using microcontact printing and lFN. Reprinted with permission from J. Foley et al., Langmuir 21,
11296 (2005). Copyright 2005 American Chemical Society.
capillary action on wetting tissue was used to generate flow, and the silicon lFN was hydrophi-
lized by gold-layer deposition, followed by PEG coating (HS-PEG) to reduce background signal
resulting from nonspecific protein binding.27 CRP and cardiac markers (i.e., myoglobin and car-
diac troponin I) were physisorbed on a PDMS slab and detected using the same sandwich im-
munoassay format. Nevertheless, preventing leakage of reagents through neighboring channels
(i.e., cross-talk) has been a challenge for such reversible sealing. Finally, instead of a PDMS
slab, Delamarche and colleagues used a gold-coated silicon lFN treated with HS-PEG for pro-
tein patterning (Figure 5(c)).158 The top surface of the lFN was coated with hexadecanethiol
(HDT) to prevent nonspecific protein adsorption. Then, using deformable PDMS stamps, protein
was microcontact-printed on the bottom surface, allowing subsequent antibody based detection.
The authors observed that the HS-PEG coating promoted protein transfer from the PDMS
stamp, as well as reduced nonspecific protein binding from solution. Stability of the transferred
protein pattern on the hydrophilic HS-PEG surface could be an issue.
binding capacity and more uniform binding patterns.160 Using a printer, a hydrophobic wax pat-
tern was created on the NC membrane to confine antibody spots. Sandwich immunoassays
using catalytic silver precipitation were demonstrated in the wax-patterned device.
Alternatively, porous membranes are fabricated in situ. These membranes benefit from tai-
lored porosity and morphology, as well as localization in specific regions of a microfluidic
channel. An NC membrane was created in situ on the glass surface by Park et al.66 After silan-
izing the glass surface with OTS to form a hydrophobic SAM, an NC membrane (dissolved in
organic solvent) was spot-dried on the glass surface. Then, the glass was bonded to a patterned
PDMS chip. The enzyme b-gal was physisorbed inside the membrane. The enzyme substrate
di-b-D-galactopyranoside (FDG) was hydrolyzed to a fluorescent product and analyzed by elec-
trophoresis. Jiangs group used an electrospinning (ES) technique to create a highly fibrous
membrane as a protein-adsorption substrate in the PDMS microfluidic devices.33,136 First, the
researchers created a nanofibrous membrane using electrospinning of PC, and sandwiched the
membrane between a glass substrate and a patterned PDMS chip.33 Then, HIV Env protein was
physisorbed and detected by a primary antibody and a fluorescein isothiocyanate (FITC) conju-
gated secondary antibody. Compared with a track-etched polycarbonate (TEPC) membrane hav-
ing a uniform pore size, the nanofiber membrane showed higher binding capacity. Similarly, a
PVDF nanofibrous membrane was created for a similar PDMS-glass slide device.136 After
adsorbing antibodies in the PVDF membrane, a multiplexed immunoassay was performed. The
study reported that the protein adsorption capacity of the PVDF membrane was 8 times larger
than that of TEPC membrane owing to an increased surface area.
Recently, paper (e.g., cellulose membrane) has drawn attention in the microfluidics
community103105,138 owing to low chip material and manufacturing costs. Intermolecular forces
including electrostatic and hydrophobic interactions are involved in protein adsorption to paper.161
Tan et al. reported a paper-PDMS glucose sensor.36 Enzyme GOx (glucose oxidase) was adsorbed
to Whatman filter paper, then a glucose solution was flowed through a PDMS microfluidic channel
to be converted into hydrogen peroxidase, which was later detected electrochemically. The stability
of GOx observed in paper was excellent showing a 2.7% RSD (relative standard deviation) in
repeatability and a one-month shelf life.
materials have been explored. Wu et al. created titania and alumina sol-gels on a sandwiched
PDMS microfluidic device (Figure 6).64 First, PDMS was treated with oxygen-plasma to gener-
ate silanol group. Then, titania sol and alumina sol were prepared by heating tetrabutyl titanate
and aluminum isopropoxide in solvent. After adding trypsin to the sol, the PDMS microfluidic
channel was filled with the sol. The silanol groups on the plasma-treated PDMS surface cova-
lently anchored the hydroxyl group of the sol by condensation, such that a stable sol-gel formed
on the PDMS surface with trypsin encapsulated within the sol-gel. BSA was digested, and pep-
tides were analyzed by MS. A faster digestion time and longer enzyme lifetime were observed,
as compared to those of a homogeneous (solution phase) reaction. Baohong Lius group
employed a similar strategy for enzyme encapsulation using nanozeolite.99 The Liu groups
microfluidic device was fabricated using thermally laminated poly(ethylene terephthalate) (PET)
sheets patterned via photoablation. Then, PSS polyelectrolyte was adsorbed to the PET surface
yielding a negative surface charge. A layer-by-layer assembly technique59 was subsequently
employed to build three layers of electrostatically combined polyelectrolyte PDADMAC (posi-
tively charged) and nanozeolite colloid crystal (negatively charged, 80 nm diameter). Finally, tryp-
sin was adsorbed to the assembled nanozeolites in order to digest BSA and protein extract from
mouse macrophage. After digestion, peptides were analyzed by MALDI-TOF MS. The zeolite
encapsulated trypsin showed more stability and faster digestion compared to free-solution trypsin.
B. Bioaffinity immobilization
The bioaffinity interaction or biospecific adsorption (Figure 2(b)) exploits specific binding
phenomena existing in nature. The bioaffinity interaction has advantages over physisorption.
A bioaffinity interaction yields relatively stronger, highly specific, and oriented protein immo-
bilization.11,106,107 Therefore, protein leakage can be minimized and immobilized protein
offers better accessibility to binding partners than random orientation strategies. Additionally,
bioaffinity immobilization can be reversed using chemical treatment, pH change, or heat treat-
ment.68,77 In most of cases, bioaffinity interactions are used in conjunction with other immo-
bilization mechanisms (i.e., physisorption and covalent bonding) with the bioaffinity reagent
used as an intermediate binding molecule between the surface and proteins. Avidin-biotin,
FIG. 6. Process of enzyme-encapsulated sol-gel inside microchannel of PDMS functionalized by oxidation in an oxygen
plasma. Reprinted with permission from H. Wu et al., J. Proteome Res. 3, 1201 (2004). Copyright 2004 American
Chemical Society.
041501-15 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
protein A/G-antibody, genetically engineered protein affinity ligands, DNA hybridization, and
aptamers have been employed in microfluidic devices.
1. Avidin-biotin
One of the most widely employed immobilization partners is avidin (6669 kDa tetrameric
glycoprotein) and biotin (water-soluble vitamin B). Avidin binds to biotin via an exceptionally
strong non-covalent interaction.164 The binding interaction is rapid and nearly insensitive to pH,
temperature, proteolysis, and denaturing agents.106 Biotin is a small molecule and conjugation
to proteins does not significantly affect protein functionality or conformation. One downside of
using the avidin-biotin system is the high cost of the proteinaceous binding reagent. NHS (N-
hydroxysuccinimide) ester is a popular commercial biotinylation reagent, which allows covalent
linking of protein amine groups with biotin. Natural avidin or engineered avidin (e.g., streptavi-
din, neutravidin, and nitrividin) can be physisorbed or covalently linked to a surface for subse-
quent immobilization of biotinylated proteins. Sometimes, avidin is attached to surfaces func-
tionalized with biotin, leaving available unoccupied biotin binding sites on the avidin for
immobilization of two biotinylated proteins.38
Owing to the popularity of the avidin-biotin immobilization strategy, streptavidin coated
PS, agarose, and glass beads are commercially available. These beads are used extensively in
microfluidic assays. Typically, proteins are incubated with streptavidin-functionalized microbe-
ads, then pseudo-immobilized in a microfluidic device using size-exclusion structures, such as
dams, weirs, and microposts. Seong and Crooks packed enzyme functionalized PS beads on a
PDMS microfluidic device with a weir structure.86 Biotinylated GOx and HRP were immobi-
lized on the streptavidin-coated PS beads. The system was used to study the mixing efficiency
of enzymatic substrate in a bead-packed microfluidic channel. Wang and Han adapted a nano-
fluidic pre-concentrator to concentration of the fluorescent proteins GFP (green fluorescent
protein) and R-PE (R-phycoerythrin). A bead-based immunoassay was employed to detect the
concentrated proteins.87 Biotinylated GFP and R-PE antibodies were attached to commercial
streptavidin-coated PS beads. The bead was then pseudo-immobilized by a microfluidic weir.
An impressive 50 pM to sub 100 fM detection limit was observed.
2. Protein A/Gantibody
Protein A and protein G are both popular antibody (IgG) immobilizing reagents, extracted
from bacteria. Protein G is known to have a wider immunoreactivity to mammalian IgGs than
protein A. Protein A or G specifically binds to the constant Fc region of IgG. Thus, the variable
Fab region of IgG is accessible to antigen binding.11 A key consideration is the orientation of
protein G or A, so that bound IgGs are away from the immobilization surface and accessible to
antigen binding. Like the avidin-biotin system, a spacer that links proteins to the planar surface
is frequently used in conjunction with protein A/G.106 An additional benefit of using protein
A/G is that antibody can be detached by acid treatment and the surface made reusable.68
GST tagging approach are: (1) cost and time associated with producing recombinant proteins
and (2) irrelevance to endogenous proteins.
4. DNA hybridization
Specific hybridization of single-strand DNA (ssDNA) with the complementary DNA (cDNA)
has been employed to immobilize proteins as well (i.e., DNA-directed protein immobiliza-
tion).25,26,77 Oligonucleotide hybridization is exceptionally stable and selective.106 Protein is first
coupled to ssDNA and then ssDNA that is linked to the protein is hybridized to the surface where
complementary ssDNA are immobilized. An ssDNA tag may be attached to the protein via cova-
lent linkage or a biotin-streptavidin linkage.106 The spatially encoded DNA-directed protein
immobilization is originated from microarray technology.166,167 Immobilization of multiple anti-
bodies and multiplexed microfluidic immunoassays can be performed using the DNA-encoded
immobilization method.25,26 Additional advantages are (1) hybridized DNA acts as a spacer arm,
and (2) immobilization is reversible by temperature control or alkaline denaturation.77
5. Antibody
Regardless of the high cost, variable affinity, and short shelf life, an antibody is a ubiqui-
tous biomolecule for immobilizing proteins. The wide-spread use of antibodies arises from the
exceptional specificity toward binding partner (i.e., immunoadsorption). Sandwich immunoas-
says in which immobilized antibody captures a target antigen are a workhorse bioanalytical
assay.168 Microbeads coated with antibody having immunoreactivity toward an IgG of a specific
animal species are commercially available. Shin et al. created a microfluidic SPE (solid phase
extraction) device for improved immunoassay sensitivity.31 In this study, PS beads coated with
goat anti-mouse IgG were incubated with mouse anti-CRP IgG. Then the beads were pseudo-
immobilized onto a frit structure of a hybrid glass substratePDMS device. Immobilized CRP
was eluted by an acidic 0.1 M glycine buffer (pH 1.8) treatment and the fluorescence signal
was detected by a photodiode integrated in the microfluidic device. Competitive immunoassays
were demonstrated.
6. Aptamers
Aptamers are oligonucleotide bioaffinity capture reagents that have drawn significant atten-
tion. Aptamers that show the highest affinity toward a target protein are selected from a synthetic,
combinational nucleotide library called SELEX (systematic evolution of ligands by exponential
enrichment).169 Aptamers are smaller than antibodies, thus a higher density of capture agents can
be coated on surfaces to yield a large binding capacity for target proteins. Compared to antibodies,
in particular polyclonal antibodies, aptamers are produced in vitroeliminating the need for ani-
mals in production.170,171 In addition, aptamers are touted to have a longer shelf life and less sensi-
tivity to environmental change. For a detailed review of aptamers in microfluidics, readers are
directed to the paper by Xu et al.170 and Mosing and Bowser.171 For aptamers for protein immobi-
lization, readers are directed to the review by Nakanishi et al.107
Yang et al. used aptamer-functionalized magnetic beads in a multilayer PDMS microfluidic
device to complete a CRP immunoassay.30 A CRP-specific aptamer sequence was screened
using SELEX, followed by biotin conjugation. Aptamers were attached to commercial
streptavidin-coated magnetic beads. After high-sensitivity, CRP (hs-CRP) was captured by
aptamers, an acridinium-ester conjugated CRP antibody was introduced for chemiluminescence
detection. Tennico et al. used a similar bead system (streptavidin-coated magnetic beads and bi-
otinylated aptamer) to detect thrombin in a sandwich immunoassay format.90 They showed that
aptamers have negligible affinity toward prothrombin and HSA, which are proteins similar to
thrombin. The simple microfluidic device consisted of PMMA (or PC) top and bottom layers,
which sandwiched an intermediate double-adhesive tape layer housing the microfluidic channel.
Two aptamers having affinity toward different epitopes of thrombin were employed as detection
041501-17 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
and capture probes in the sandwich immunoassay, whereas antibody is commonly used as the
detection probe in aptamer-based immunoassays.30
C. Covalent bond
Covalent bonds are a frequently used immobilization mechanism in microfluidic assays
(Figure 2(c)).11 The immobilization surface is activated via reactive reagents. The activated sur-
face reacts with amino acid residues on the protein exterior and forms an irreversible linkage.
One tends to rely on covalent immobilization if high, stable protein coverage is required.
Bifunctional spacer molecules are a common approach to forming an irreversible bond between
proteins and the immobilization surface. In such an approach, one end of a spacer molecule is
covalently linked to an activated surface, and then a protein is covalently linked to the other
end of the spacer. Alternatively, another spacer or protein capture agent (e.g., streptavidin) is
crosslinked on the other end. Unreacted active functional groups are blocked or deactivated
(e.g., BSA,106 hydroxylamine,83 ethanolamine,172 or lysin40). Disadvantages of covalent linkage
include reduced activity of proteins (by forming linkage on active sites),106 toxic reagents,125
and complicated chemistry.125 A covalent bond can be formed on active sites of proteins,
resulting in reduced activity.11 The covalent attachment reaction is that the reaction is usually
slow, so that the protein and surface require long incubation times of hours up to a full day
(e.g., epoxide61). Therefore, covalent linking is usually performed as a preparatory step before
performing microfluidic assays. An enormous variety of covalent conjugation chemistries are
available. In this review, only a small subset of conjugation chemistries employed especially in
microfluidic devices is introduced. For more information beyond the present scope, refer to a
review by Feijen et al.106 and the excellent reference by Hermansson.125
1. Amineglutaraldehydeamine
Amino groups (-NH2) of lysine are the most common covalent binding sites because lysine
residues are usually present on the exterior of proteins. Aldehyde is a reactive compound that
forms the labile Schiff base with the amine and can be further reduced to form a stable second-
ary amine bond using NaCNBH3 or NaBH4. Glutaraldehyde (GA) is a bis-aldehyde compound
that has two reactive ends. GA can crosslink two amine functional groups, for example,
between two proteins or between a protein and a surface-immobilized polymer with amine
groups (e.g., PEI). The Schiff bases formed on proteins are stable without further reduction by
NaCNBH3 or NaBH4;125 and indeed, a large number of protein immobilization strategies that
rely on GA in microfluidic devices are completed without further reduction. When used with a
glass or a silicon surface, aminosilane compounds like APTES [(3-aminopropyl)triethoxysilane]
are used because they can bind to hydroxyl functional groups of glass at one end and possess
an amine functional group to facilitate covalent linkage with proteins on the other end.117 GA
had been extensively used to form stable protein microarrays on glass substrates.173 Regnier
et al. used APTES to coat wet-etched glass microfluidic channels with aminosilane.174 Then,
GA was electroosmotically injected for 2 h and the enzyme b-gal was covalently immobilized
on the glass channel walls. Porous-silicon micropits were treated with APTES and GA for tryp-
sin immobilization by Marko-Vargas group.60 The Schiff base was further reduced by
NaCNBH3 for stable bonding. Digested proteins were analyzed by MALDI-TOF/MS. Wang
et al. used a similar approach in immobilizing proteins and antibodies in a device consisting of
a silicon substrate and a PDMS microfluidic array.111 Ellipsometry was used for label-free
microarray immunoassays. Richter et al.91 used the same chemistry to immobilize enzymes
xanthine oxidase (XOD) and HRP on porous silica beads. After trapping the silica beads behind
a weir structure, they successfully detected hydrogen peroxide in a glass microfluidic device.
As described earlier, proteins are usually physisorbed to polymer membranes (e.g., NC, PS,
and PVDF) clamped in a microfluidic device. Kitamoris group immobilized enzymes onto an
in situ fabricated nylon membrane using covalent bonding (Figure 7).100 First, a glass microflui-
dic channel was treated with APTES and then a vertical nylon membrane was fabricated by
interfacial polycondensation. Co-injecting immiscible laminar flows of adipoyl chloride in
041501-18 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 7. In situ polymerized nylon membrane for an enzyme assay. (a) In situ polymerization of nylon membrane using or-
ganic/aqueous two-phase flow in an X-shaped microfluidic chip, and (b) TLM (thermal-lens microscopy) was used to detect
product and substrate. Reprinted with permission from H. Hisamoto et al., Anal. Chem. 75, 350 (2003). Copyright 2003
American Chemical Society.
1,2-dichloroethane and hexamethylenediamine (HMD) in 0.1 M NaOH created 10-lm thick nylon
membrane. Amino groups on the membrane were activated by GA, followed by immobilization of
HRP and further reduction of the Schiff base using NaBH4. The activity of HRP was tested by
introducing substrate on one side and hydrogen peroxide on the other side of the membrane.
Hydrogen peroxide diffused through the membrane and HRP generated a colored product.
Yu et al. used an intermediate PVA [poly(vinyl alcohol)] layer to minimize nonspecific
protein adsorption to PDMS.175 The researchers silanized an oxygen-plasma-treated PDMS sur-
face with APTES. In order to form the hydrophilic layer, the amine group on APTES was acti-
vated with GA to attach PVA (via hydroxyl group of PVA176) to PDMS. Then, GA was used
once more to covalently link proteins such as IgM, BSA, and IgG to the PVA layer. A sand-
wich immunoassay was demonstrated. Compared to the native PDMS surface, the SNR (signal-
to-noise ratio) was improved owing to low nonspecific adsorption and high antibody binding
capacity. Thomsen et al. took an alternative approach to functionalizing a PDMS surface lack-
ing functional groups. These authors applied pyrogenic silicic acid (i.e., silicon oxide powder
prepared in oxyhydrogen flame) to PDMS, so that the PDMS surface exhibited hydroxyl
groups.52 Using the added hydroxyl group and APTES, then activating with GA, the enzyme
CelB (b-glycosidase) was immobilized to hydrolyze lactose to glucose and galactose.
Another aminosilane coupling reagent, 3-aminopropyl)trimethoxysilane (APTMS),120 is
also popular because of better reactivity than APTES. Glass et al.117 provide a review of vari-
ous organosilanes and deposition techniques of the organosilanes. A spacer of APTES or
APTMS may be too short to properly immobilize large proteins (such as antibodies and
enzymes) to planar channel surfaces in high capacity owing to steric hindrance. The scheme
may also cause reduced activity owing to partial physisorption to channel surface.40 Therefore,
a hydrophilic polymer matrix such as dextran (DEX),29,68 chitosan,130 and PEG40,120 are often
used as longer spacers for improved assay sensitivity.
041501-19 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 8. Zero-powered COC immunoassay chip with patterned micropillars for covalent antibody attachment. The COC sur-
face is oxidized in oxygen plasma and silanized with APTES. The resultant amino terminated surface is subsequently func-
tionalized with a dextran matrix. Finally, antibody is immobilized via Schiffs base coupling to the dextran matrix.
Adapted from C. J onsson et al., Lab Chip 8, 1191 (2008) with permission from The Royal Society of Chemistry.
041501-20 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
protein-friendly environment, and an enlarged surface area for a high antibody binding capacity.
Baeza et al. took an in situ aldehyde generation approach in order to immobilize enzyme in
LTCC (low temperature cofired ceramics) microfluidic device.54 A commercial agarose bead
was activated to have a glyoxal functional group (agarose-O-CH2-CHO) by etherification with
2,3-epoxypropanol and oxidation with NaIO4.128 The aldehyde functional end of glyoxal aga-
rose was used to immobilize b-gal after the Schiff-base reduction with NaBH4. The beads were
pseudo-immobilized using microcolumns in a reaction chamber. The enzyme activity was meas-
ured colorimetrically with enzyme stability observed for 6 months.
3. AmineNHS (N-hydroxysuccinimide)
Probably, the most commonly used covalent linking agent is NHS ester.106 NHS ester
reacts with amines on proteins and yield stable amide bonds while releasing NHS leaving
groups.125 Sulfo-NHS (N-hydroxysulfosuccinimide) is preferred over NHS because sulfo-NHS
is more water soluble while showing the same reactivity and specificity as NHS. Delamarche
et al. provided an early report of protein immobilization in microfluidic device156 by introduc-
ing a PDMS microfluidic network (lFN) comprised of PDMS irreversibly bound to glass or sil-
icon. The surface of the glass or silicon wafer was coated with aminosilane. Amine functional
groups of the aminosilane were activated by the NHS ester for antibody immobilization.
Paleceks group used PEG hydrogel micropillars copolymerized with an acryloyl-functionalized
NHS ester for microfluidic kinase activity assays and immunoassays.82,83 The microfluidic-chip
fabrication process is rather interesting in that the authors fabricated a microfluidic channel
using in situ polymerization of the monomer isobornyl acrylate (IBA) in an empty chamber
made of a bottom glass slide, a polycarbonate top, and an intermediate adhesive rim, whereas
typical microfluidic chip fabrication is done by etching bulk material.133 After the glass slide
was silanized with (3-acryloxypropyl)-trimethoxysilane to provide an acryloyl group, macropo-
rous PEG pillars were photopolymerized using PEGDA monomer. PEG porogens of various mo-
lecular masses were used to form macropores. A macroporous structure is favorable over a
nanoporous gel for incorporating large proteins such as antibodies and kinase. The 6-((acryloyl)-
amino)hexanoic acid NHS ester was copolymerized in the hydrogel pillars for covalent immobili-
zation of proteins such as GST-fused GFP and GST-fused CrkL (Crk-like) protein. GST antibody
and kinase activity assays (phosphorylation) of K562 cell lysate were demonstrated.83
Characterization of diffusion of 250 kDa dextran and GST-GFP showed improved mass transport
through the gel as compared to nanoporous PA or PEGDA hydrogels.82
5. CarboxylateEDC NHSamine
NHS esters can be formed on-demand using carbodiimide (e.g., EDC), NHS, and carboxy-
lates to immobilize proteins.177 Didar et al. created multiplexed protein microarrays in a micro-
fluidic device consisting of a glass substrate and PDMS microchannels after microcontact printing
of APTES spots.177 NHS and EDC were used to covalently link proteins to the amine group of
APTES. Carboxylate groups of antibodies were linked to the end of APTES after releasing the
NHS leaving group. Antibodies for CD34, CD31, and CD36 proteins were immobilized on the
patterned APTES spots and multiplexed sandwich immunoassays were demonstrated. Hu et al.
used EDC and NHS chemistries to immobilize detection antibody to aqueous quantum dots
041501-21 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
(aqQD) for microfluidic sandwich immunoassays.178 Usually, high quantum-yield QDs are syn-
thesized in an organic phase, which requires post treatment for water solubility. In contrast, the
CdTe core/CdS shell QD was prepared in an aqueous phase.179 The QD was covered with 3-
mercaptopropionic acid (MPA) in order to yield a carboxylic-acid functionalized surface. The car-
boxylic group was later activated with the NHS ester, and then covalently linked to the amine
groups of antibodies. Capture antibody was covalently attached to commercial silanized glass
slides, against which the PDMS microchannels were sealed. Sandwich immunoassays of the can-
cer biomarkers carcinoma embryonic antigen (CEA) and a-fetoprotein (AFP) were demonstrated
using this approach.
6. Amine/sulfhydrylepoxide
Epoxides form covalent bonds with primary amines at mild alkaline pH or with sulfhydryl
groups (-SH) in the physiological pH range.125 The advantages of using epoxides include a sim-
ple protocol, a neutral pH range, and relevance to aqueous conditions. Due to slow kinetics,
protocols suggest using high concentrations of epoxide and additional functional groups to
promote the adsorption of proteins.106 A common epoxide functional group used in protein
immobilization is glycidyl.96,172,180,181 Thierry et al. reported epoxide-based capture of
tyrosine-kinase human epidermal growth factor receptor (HER2) on PDMS microfluidic devi-
ces.181 They coated a PDMS microchannel and the surface of a glass substrate using a pulsed
plasma polymerization of allyl glycidyl ether (AEG) monomer (Figure 9).172 The authors rea-
soned that a common epoxidation reagent GOPTS (3-glycidoxypropyltrimethoxysilane) was
unstable owing to hydrolysis, but pulsed plasma yielded a conformal, adherent, defect-free gly-
cidyl coatings on the surface.172 Herceptin, a commercial HER2 antibody, was attached to the
coating via the epoxide group of the AEG polymer allowing capture of CTCs (circulating tumor
cells). The authors also tested a multilayer coating consisting of AEG polymer and PEGDA de-
posited via epoxide crosslinking. The PEGDA layer was activated with NHS and EDC to im-
mobilize Herceptin. In comparison to the AEG-only coating, reduced nonspecific protein
adsorption was observed but lower Herceptin density was obtained.
7. Amineisothiocyanate
The reaction of an aromatic amine with thiophosgene (CSCl2) yields isothiocyanate
(-NCS), which forms a stable bond with primary amine groups.125 Sui et al. used the isothiocy-
anate chemistry to immobilize proteins, peptides, and DNA on the PDMS surface of a glass-
PDMS microfluidic device (Figure 10).120 The approach used solution-based oxidation of
PDMS, not common plasma-based oxidation, owing to better stability of the oxidized surface.
The PDMS channel was oxidized with H2O2 and HCl, then silanized with 2-[methoxy(polyethy-
leneoxy)propyl] trimethoxysilane to graft PEG onto the PDMS, followed by APTES treatment
FIG. 9. Pulsed plasma epoxidation of PDMS surfaces and bioconjugation of proteins. Reprinted with permission from B.
Thierry et al., Langmuir 24, 10187 (2008). Copyright 2008 American Chemical Society.
041501-22 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 10. PEG-grafted PDMS surface for biomolecule immobilization. (4) Preparation of the PEG-grafted PDMS micro-
channels and (6) amine-grafted PDMS microchannels. The amine-grafted microchannels can be activated by thiophosgene
to obtain (7) the isothiocyanate-grafted PDMS microchannels as a precursor for (8) the RGD-grafted, (9) DNA-grafted, and
(10) PSCA-grafted PDMS microchannels. Reprinted with permission from G. Sui et al., Anal. Chem. 78, 5543 (2006).
Copyright 2006 American Chemical Society.
for amination. Subsequently, thiophosgene was introduced to convert amine groups to reactive
isothiocynate groups. Later, in situ generated isothiocynate reacted with amine-terminated
PSCA (prostate stem cell antigen) protein, tripeptide RGD (arginine-glycine-aspartic acid), and
aminated DNA for immobilization. Sandwich immunoassays of PSCA, DNA hybridization, and
RGD-mediated cell adhesion were demonstrated. The authors found that PEG was effective in
repelling proteins from a surface for more than 2 months.
8. Amineazlactone
Azlactone is formed by the cyclization of N-acyl-a-amino acids. Azlactone readily reacts
with nucleophiles such as amines and thiols at room temperature to form amide bonds.182
Peterson et al. reported a microfluidic protein digestion device using a photopatterned polymer
monolith where trypsin was immobilized using azlactone.61 Glass chips were silanized with 3-
(trimethosysilyl)propyl methacrylate, leaving the methacryloyl group to covalently link polymer
monolith to the glass substrate.183 Azlactone functionalized monomer (2-vinyl-4,4-dimethyla-
zlactone ethylene dimethacrylate acrylamide or 2-hydroxyethyl methacrylate), porogenic
solvent (dodecanol, decanol, or its mixtures with cyclohexanol), and photoinitiator (2,2-dime-
thoxy-2-phenylacetophenone) were used for UV polymerization of the monolith. After trypsin
was covalently immobilized on the azlactone-functionalized surface, unreacted azlactone was
041501-23 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
blocked by ethanolamine. Casein was digested and analyzed by MALDI-TOF/MS. The reaction
time was 10 s, whereas in-solution digestion required 10 min.
9. Aminep-nitrophenyl ester
p-nitrophenyl ester is reactive to amines across the slightly basic pH range spanning 79.
The ester forms stable amide bond with proteins.125,184 The p-nitrophenyl ester shows a similar
reactivity to the NHS ester and is frequently used as one end of heterobifunctional linker (e.g.,
p-nitrophenyl iodoacetate and biotin-4-nitrophenyl ester).
11. Sulfhydrylmaleimide
Maleimide is a popular reagent for the crosslinking of sulfhydryl groups. Thus, maleimide
is used to form covalent links with the cysteine residues of proteins. Maleimide reactions with
thiols are specific at pH 6.57.5. However, maleimide also reacts with amines at higher pH val-
ues.106 Alkylation reaction of the maleimide double bond with sulfhydryl forms stable thioether
bonds.125 Maleimide is often used as one end of a heterobifunctional cross linker such as sulfo-
SMPB (sulfosuccinimidyl 4-[p-maleimidophenyl]butyrate).187 Hydrolysis in aqueous solution
may present issues.
1. Physisorptioncovalent bond
Yakovleva et al. studied five different polymer coatings on porous silicon surfaces40
(Figure 11). In the first system, porous silicon was treated with APTES, then with GA for anti-
body immobilization. Unreacted GA was blocked by L-lysine. In the second system, BPEI
(branched PEI) was directly adsorbed on the porous silicon surface by electrostatic interaction
041501-24 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 11. Structures of the five tested antibody functionalized porous silicon surfaces. Reprinted with permission from J.
Yakovleva et al., Anal. Chem. 74, 2994 (2002). Copyright 2002 American Chemical Society.
between negatively charged silicon and positively charged PEI. Amine groups on BPEI were
activated with GA and antibody was immobilized. In the third system, porous silicon was
epoxide-functionalized with GOPTS. The amine groups of BPEI were first linked to GOPTS,
then the same procedure used in the second system studied was employed to immobilize anti-
body. In the fourth system, positively charged LPEI (linear PEI) was physisorbed onto a silicon
surface by electrostatic interaction. Then, the same procedure used in the second system studied
was applied to immobilize antibody. Finally, in the fifth system, LPEI was physisorbed on a sil-
icon surface, then antibody also physisorbed to the LPEI surface. The authors observed that the
fourth system showed the best sensitivity and antibody reliability, better than direct immobiliza-
tion. The authors claimed that, owing to the long chain of LPEI and BPEI, antibody was situ-
ated away from the silicon surface, and covered the silicon surface effectively. The PEI layers
also prevented nonspecific protein adsorption and provided more antibody binding sites to anti-
gens than the bare porous silicon surface.
As a native surface of PS, COC, and PMMA lacks a ligand for covalent linkage, the surfa-
ces were modified with various methods. Besides common oxygen-plasma treatment, one
approach to surface coating has been used as an intermediate polymer layer that has an attrac-
tive interaction with the surface for further protein immobilization. Another approach has been
to forcibly introduce a ligand to the plastic. Bai et al. performed a study of protein adsorption
on PMMA microfluidic chips.78 The PMMA chips were first treated with 1 M NaOH for hydro-
philization, and then coated by physisorption of BPEI, PAH, and HMD. The amine functional
group on the coatings was then activated by GA for antibody covalent immobilization. After
blocking by BSA, sandwich immunoassays were performed. Among bare PMMA and BPEI-,
PAH-, and HMD-treated PMMA surfaces, the signal was highest for the BPEI-treated PMMA.
041501-25 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
The authors attributed the result to the spacer effect for keeping the antibody away from the
hydrophobic surface thereby minimizing steric hindrance to immunobinding.
3. Physisorptionbioaffinity immobilization
Schult et al. exploited the hydrophobicity of PMMA to physisorb neutravidin, then immo-
bilized a biotinylated monoclonal capture antibody.37 hCG (pregnancy hormone chorionic) was
detected by sandwich immunoassay. The approach did not use any blocking, but showed insig-
nificant nonspecific protein adsorption. Linder et al. used three layers of biopolymer (biotinyl-
ated IgG, neutravidin, and biotin-conjugated dextran, in order of coating) to prevent nonspecific
binding of protein on the PDMS surface and to introduce surface charge for cathodic electro-
osmotic flow.191 For immunoassays, biotin-conjugated antibody was attached to the neutravidin
layer instead of the dextran. The authors observed that nonspecific binding was significantly
reduced in the composite coating. However, using costly IgGs in surface coating may not be
practical. Wen et al. employed a complex multilayer approach to immobilize antibody to
PMMA surfaces (Figure 12).80 First, they prepared two positively charged linkers PLL-g-PEG-
biotin and PLL-g-PEG using graft copolymerization. Then, the PMMA surface was treated with
oxygen plasma to become hydrophilic, and then acrylic acid was UV graft polymerized on the
PMMA surface. This process resulted in a polyacrylic-acid coated PMMA surface that was
FIG. 12. Schematic illustration of the stepwise process involved in the biotin-PLL-g-PEG and protein A-based antibody
immobilization. Reprinted with permission from X. Wen et al., J. Immunol. Methods 350, 97 (2009). Copyright 2009
Elsevier.
041501-26 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 13. Fabrication of an r-SBM for SEB immunoassay in a PDMS microfluidic chip. (a) Streptavidin reinforced SBM,
(b) surface functionalization with biotinylated anti-SEB IgG, (c) capture of SEB, and (d) SEB-antibody binding followed
by incubation with fluorescently labeled secondary antibody to generate a signal. A top view of the microchannel is also
shown. Reprinted with permission from Y. Dong et al., Lab Chip 6, 675 (2006). Copyright 2006 The Royal Society of
Chemistry.
FIG. 14. (a) Sequence-specificity control after immobilization of two different receptor sequences (A and B) onto the COC
surface. First, Cy5-labeled oligonucleotide complementary to sequence A and afterward rabbit-IgG conjugated oligonu-
cleotide complementary to sequence B were hybridized. Reprinted with permission from H. Wang et al., Electrochem.
Commun. 12, 258 (2010). Copyright 2010 Elsevier. (b) Optical micrographs of double-line bead micropatterns and sche-
matic illustration of the sandwich immunocomplex formation on the streptavidin-coated bead micropattern. The micro-
channel is filled and incubated, subsequently, with (i) biotin-labeled capture antibody, (ii) target antigen, and (iii) Cy3-
labeled detection antibody, forming the sandwich immunocomplex. Reprinted with permission from V. Sivagnanam et al.,
Anal. Chem. 81, 6509 (2009). Copyright 2009 American Chemical Society. (c) Schematic illustration of the stepwise pro-
cess involved in the TR-catalyzed protein A-based antibody immobilization. Reprinted with permission from Y. Yuan
et al., Biotechnol. Bioeng. 102, 891 (2009). Copyright 2008 Wiley InterScience.
nanoparticles for thrombin capture. Thrombin was injected, and subsequently an ALP-
functionalized detection aptamer was introduced. Finally, the enzyme ALP converted the sub-
strate 4-aminophenyl phosphate to the product p-aminophenol, which was amperometrically
detected. The authors reported a 1-pM detection limit. Sivagnanam et al. patterned antibody
functionalized microbeads on a glass surface92 using dot and line patterns of APTES. The
APTES pattern was created using a lift-off process on the glass substrate of a hybrid glass-
PDMS microfluidic device (Figure 14(b)). Owing to the use of a lift-off process, the APTES
patterns have ridges. Amine groups on APTES are positively charged while streptavidin is neg-
atively charged at the pH of the solution (7.4). The authors observed an exceptionally strong
electrostatic interaction between the APTES pattern and the beads, so strong, in fact, that the
beads withstood the shear force of hydrodynamic flow and magnetic force, and did not lift off
of the surface under the conditions studied. With this assay, mouse IgG was detected down to
041501-29 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 15. Smart immobilization methods for reproducible, high-sensitivity, multiplexed, high-throughput microfluidic
immunoassays, or enzyme assays. (a) Photoactivated protein immobilization, (b) electrochemically activated immobiliza-
tion, (c) thermally activated immobilization, (d) photoactivated protein elution, (e) spatially addressable multiplexed pro-
tein immobilization, (f) reversible protein immobilization for repetitive assay, and (g) multifunctional immobilization
surface for simplified immobilization procedure.
conjunction with precise and automated fluid-handling capability and enclosed structure (i.e.,
better protection from environment compared to microarrays), multiplexing capability was
sought in microfluidic devices. Thus, we introduce DNA-directed immobilization for multi-
plexed (i.e., multiple proteins25) and multiparameter (i.e., multiple kinds of biomolecules26)
analysis (Figure 15(e)). Unless designed to be disposable, a microfluidic device has to be
cleaned for reuse, and the cleaning was usually done under harsh condition (e.g., strong oxidiz-
ing agent at high temperature46). We introduce a smart approach to refresh an immobilization
surface in mild condition for repeated use of microfluidic chips (Figure 15(f)).63 Finally, a com-
plicated multiple-step procedure is usually required to introduce various functions on the immo-
bilization surface (e.g., anchoring to the substrate surface, repelling unwanted biomolecule, and
immobilization target proteins). Thus, we introduce smart single composite coatings that can
perform these various functions (Figure 15(g)).
A. Photoactivated immobilization
Spatial control of protein patterning in a microfluidic device is beneficial to multistep, mul-
tiplexed immunoassays21,22 or enzyme reactor design.95,201 Microcontact printing is a primary
method for protein patterning with spatial control, but the method does have constraints as pat-
terning should be done before assembly of the microfluidic chip.201 Introducing heterogeneous
surface chemistries and immobilizing multiple proteins for multiplexed and multistep assays in
a completely assembled microfluidic device, photopatterning through a high resolution photo-
mask is considered most promising.96,195,202 Microfluidic chips are usually transparent (at least
one side), so the UV light can penetrate the substrate and photo-initiate protein immobilization.
Unlike an electrochemical method, another patterning approach, photoactivated immobilization
does not require microfabrication of electrodes.130,195
1. Physisorption
Zhan et al. photopatterned PEG hydrogel patches to physically encapsulate enzymes in
microfluidic devices consisting of a PDMS substrate and a glass slide.95 First, the glass surface
was silanized with TPM [3-(trichlorosilyl)propyl methacrylate], and then PEGDA was photopat-
terned with the photoinitiator HOMPP (2-hydroxy-2-methylpropiophenone). GOx and HRP
041501-31 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
were encapsulated in PEG hydrogels having 1 nm size pores. After the first PDMS microchan-
nel device was detached from the glass slide, a second PDMS microchannel device with larger
width and depth than the first PDMS microchannel was placed to direct fluid flow over and
around the patterned hydrogel patches. Glucose penetrated the PEG hydrogel and was detected
by GOx-HRP in a two-step enzyme assay. In addition to the enzyme assay, the researchers
incorporated a pH-sensitive dye (SNAFL) to measure the pH. Using photopatterning, multiple
patches of hydrogel each encapsulated with a different enzyme were created in a single channel
to offer a multiplexed enzymatic assay. Koh and Pishko reported a similar PEGDA gel patch
for enzyme entrapment to study enzyme kinetics (ALP and urease).57
2. Bioaffinity interaction
Cremers group introduced an enzyme photopatterning method in a PDMS microfluidic de-
vice using photobleaching of fluorophore-conjugated biotin.201 Bovine fibrinogen was used to
passivate the surface of a glass cover slide because the fibrinogen showed a better enzyme bind-
ing density and lower nonspecific protein adsorption than BSA, the commonly used blocking
material. A commercial fluorophore-conjugated biotin solution was introduced and a 488 nm
laser (300 lm beam diameter) irradiated the channel. The light created singlet oxygen, which
subsequently attacked the fluorophore to generate free radicals for crosslinking of biotin to fibri-
nogen.196 A set of streptavidin-conjugated enzymes (GOx, ALP, and HRP) was immobilized in
a single channel and the enzyme turnover number was estimated (Figure 16(a)). Castellana
et al. reported UV-light-induced silver nanoparticle patterning and selective protein capture on
the pattern.42 A TiO2 film was deposited on a glass wafer, and then the wafer was sealed with
a PDMS microfluidic channel (Figure 16(b)). A unique property of TiO2 is that it creates holes
FIG. 16. (a) Schematic diagram of the photoimmobilization process of enzyme. (1) Passivation of the surface with a fibri-
nogen monolayer is followed by (2) biotin-4-fluorescein surface attachment. This is accomplished by photobleaching with
a 488-nm laser. (3) Next, the binding of streptavidin-linked enzymes that can be exploited to immobilize catalysts and (4)
monitor reaction processes on-chip. Reprinted with permission from M. A. Holden et al., Anal. Chem. 76, 1838 (2004).
Copyright 2004 American Chemical Society. (b) Schematic diagram for the protein patterning using a silver nanoparticle
film. First, an AgNO3 solution is introduced into the microchannel. Next, UV radiation is passed through a photomask onto
the backside of the TiO2 thin film. Ag ions adsorbed at the interface are selectively reduced by photoelectrons, which
grow into nanoparticle films. Thiol chemistry was used to immobilize streptavidin. Reprinted with permission from E. T.
Castellana et al., Anal. Chem. 78, 107 (2006). Copyright 2006 American Chemical Society.
041501-32 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
and electrons upon UV exposure. The electrons were used to drive electrodeposition of silver
after the introduction of a AgNO3 solution and UV irradiation through a photomask. The
researchers used this approach to generate various patterns of silver nanoparticles. A mixture of
biotin-PEG-disulfide and PEG-propionate-disulfide was introduced to functionalize the silver
pattern with biotin and PEG (i.e., antibiofouling molecules) via thiol chemistry. DNP (N-2,4-
Dinitrophenyl)-PEG-disulfide was functionalized on another silver pattern. Streptavidin binding
to the biotin-functionalized silver pattern and anti-DNP antibody binding to the DNP-
functionalized silver pattern were demonstrated.
3. Covalent immobilization
Wang et al. embedded PAA on a PDMS surface by photopatterning using surface dif-
fused benzophenone.119 PDMS was submerged in acetone to cause swelling of the material, so
that benzophenone could penetrate into the swollen PDMS. With acrylic acid monomer and UV
exposure, PAA was formed as deep as 50 lm underneath the surface (Figure 17(a)). PAA was
tightly anchored to the PDMS surface, and carboxyl functional groups on PAA were further
activated with EDC in order to covalently bind antibody directly or to bind antibody through
immobilized protein A. As protein A can bind up to four antibodies, fluorescence signal was
improved 3 folds over directly attaching antibody to the surface without protein A. Fiddes
FIG. 17. (a) Cross-linking of proteins to PDMS micropatterned with PAA. The PDMS is photopatterned with PAA. In a
subsequent step, amide bonds are formed between the carboxyl groups of the PAA and amino groups of proteins. Reprinted
with permission from Y. Wang et al., Anal. Chem. 77, 7539 (2005). Copyright 2005 American Chemical Society. (b) PAA-
grafted PDMS for protein immobilization. (1) Native PDMS, (2) PDMS with a grafted layer of PAA, and (3) PDMS with a
grafted layer of PAA that has been conjugated with FITC-casein. Reprinted with permission from L. K. Fiddes et al.,
Biomaterials 31, 315 (2010). Copyright 2010 Elsevier. (c) GMA photopolymerization and protein immobilization in a specific
region on a glass slide using glycidyl functionalized hydrogel. Adapted from K. H. Park et al., Biosens. Bioelectron. 22, 613
(2006) with permission from Elsevier. (d) Detailed schematic of how covalently attached, photografted, antibody-containing
tethers can be used to provide rapid detection of a specific antigen using a sandwich immunoassay approach. Adapted with per-
mission from R. P. Sebra et al., Anal. Chem. 78, 3144 (2006). Copyright 2006 American Chemical Society.
041501-33 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
reported protein immobilization on a PDMS surface via photografting of PA.188 Upon UV radi-
ation, benzophenone abstracts hydrogen from methyl group of the PDMS to produce radicals so
that graft polymerization of acrylamide monomer is initiated (Figure 17(b)). By activating with
EDC and sulfo-NHS, fluorescently labeled casein and collagen were immobilized on a grafted
PA layer (200 nm thickness). Various protein patterns including spots of casein, linear gradients
of casein and collagen, and multiple spots of casein, collagen, and IgG were demonstrated. The
activity of immobilized trypsin was also examined. Park et al. used an approach similar to
Crooks work95 in photopatterning a hydrogel patch to immobilize antibodies in a hybrid
PDMS-glass slide microdevice.96 Glass was silanized with TPM, and then GMA (glycidyl
methacrylate) was photopatterned with HOMPP. Anti-human IgG and anti-mouse IgG were co-
valently immobilized to a GMA patch via glycidyl (epoxide) functional groups on the surface
after a 20-min incubation (Figure 17(c)). A downside of the GMA patch is that protein is im-
mobilized on the surface and does not penetrate into the material, which may reduce assay sen-
sitivity. Sebra et al. immobilized PEG-conjugated antibodies by graft polymerization on a DTC
(dithiocarbamate)-based photoreactive polymer substrate.101 First, antibody was acrylate-
functionalized by reacting with NHS-PEG-acrylate. Thus, the acrylate is covalently attached to
the antibodies with an intermediate PEG spacer. The substrate for the microfluidic device was
made by UV polymerization of urethane diacrylate (UDA) and triethylene glycol diacrylate
(TEGDA) in the presence of tetraethylthiuram disulfide (TED) and photoinitiator 2,2-dime-
thoxy-2-phenylacetophenone (DMPA). Because of the TED, this substrate has photoreactive
carbamate for subsequent photografting reactions. PEG monoacrylate and acrylated antibody
were photografted upon UV exposure (Figure 17(d)). The hormone glucagon was detected using
a sandwich immunoassay with picomolar sensitivity. The authors claimed that antibody copoly-
merized into the PEG polymer improved the antibody activity owing to a solvated and mobile
polymer environment. They further observed reduced nonspecific protein binding.
Smart materials have also been used for photoactivated protein immobilization after protein
analysis, such as a separation step.21,22 Probed IEF and Western blot are workhorse protein-
analysis techniques comprised of multiple biochemical assays. Each assay consists of a separa-
tion step, a transfer/immobilization (blotting) step, and a probing step (with immunoreagent).
Inspired by photoactivated immobilization on a glass capillary surface,203 the two multistep
assays are realized in a simple microfluidic device (i.e., straight microchannel). Owing to sim-
plicity and the small footprint of the microfluidic device, multiple assays were integrated on a
single chip, and thus parallel analysis of multiple targets in a short duration assay was reported.
In one example, IEF and photoactivated protein immobilization were conducted, followed by
antibody probing of the separated proteins (Figure 18(a)).21 To prepare the channel to conduct
all assay stages, the surface of the glass channel was first silanized and acrylated using 3-(tri-
methoxysilyl) propyl methacrylate. Then, the benzophenone-functionalized monomer N-[3-[(4-
benzoylphenyl)formamido]propyl] methacrylamide (BPMAC, synthesized in house) and acryl-
amide monomer were polymerized via a free-radical process using the initiator system of
TEMED (N,N,N,N-tetramethylethylenediamine) and APS (ammonium persulfate). After com-
pletion of IEF, the entire separation channel was irradiated with UV light, which initiated cova-
lent bonding of proteins to the benzophenone copolymerized in the channel-filling PA gel. In
this way, the isoelectric-point-based protein separation pattern was immobilized on the PA gel.
PSA (prostate specific antigen) in human prostate cancer cell lysate and crude sera from meta-
static prostate cancer patients were analyzed via IEF, photo-immobilized, and immunostained
using fluorescently-labeled primary and secondary antibodies. Compared to immobilization of
separated proteins in a capillary-based probed IEF assay,203 the channel-filling PA gel approach
reported a 2 improvement on assay speed and a 180 improvement of protein capture effi-
ciency. Use of microfluidic design with purely electrophoretic control of material introduction
eliminated the need for fluidic components such as valves and pumps, thus simplifying the
external hardware. In following studies, Western blotting was realized in a single microfluidic
channel using a similar benzophenone-based covalent protein photoimmobilization strategy in a
3-D channel-filling gel (Figure 18(b)).22 In this assay, gel polymerization was initiated using a
photo-initiator based system. PA gel was photopolymerized inside microchannels using
041501-34 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 18. (a) Design and operation of the microfluidic IEF assay. (1) Glass microfluidic device (scale bar: 2 mm), (2) the 80-
min five-step assay is completed in a single microchannel, (3) schematic of microchannel cross-section depicting photoacti-
vated protein immobilization: analytes are electrophoresed through the PA gel, exposed to UV, and covalently immobilized
(scale bar: 5 lm), (4) schematic of reaction between polypeptide backbone and benzophenone copolymerized in the PA
gel. Ph denotes phenyl group. Reprinted from permission from A. J. Hughes and A. E. Herr, Proc. Natl. Acad. Sci. U.S.A.
109, 5972 (2012). Copyright 2012 National Academy of Sciences, USA. (b) Single-channel microfluidic Western blotting.
The microfluidic Western blotting step is comprised of: (1) analyte stacking and SDS-PAGE within the PA gel; (2) capture
of separated protein bands (blotting) onto the benzophenone-copolymerized PA gel under UV exposure; (3) electropho-
retic introduction of fluorescently labeled detection antibodies for the target analyte; and (4) standard microscope-slide-
sized chips with a scalable electrode array, accommodating 48 blots per chip in triplicate (144 microchannels). Reprinted
from permission from A. J. Hughes and A. E. Herr, Proc. Natl. Acad. Sci. U.S.A. 109, 21450 (2012). Copyright 2012
National Academy of Sciences, USA.
riboflavin 50 monophosphate, TEMED, and a blue LED light source (470 nm) to have more spa-
tial control than previous APS-based chemical polymerization.21 Riboflavin was selected, as ex-
posure of the gel to blue light does not appreciably activate benzophenone, thus preserving the
photocapture function. After high-resolution protein sizing (SDS-PAGE), proteins were immobi-
lized on the BPMAC-copolymerized PA gel using UV light. The capture efficiency was >75%,
rivaling capture of conventional Western blotting membranes. After immobilization, target pro-
teins were detected using primary antibody and ALP-conjugated secondary antibody.
Multiplexed detection of ovalbumin, trypsin inhibitor (TI), and b-gal was demonstrated. Two
biologically relevant systems were also reported: blotting of NFjB (nuclear factor kappa-light-
chain-enhancer of activated B cells) from cell lysate and detection of human antibodies against
gp120 and p24 proteins in human sera. In total, 48 parallel Western blots were demonstrated in
a single 24-minute assay, with probing for two targets per microfluidic Western blot. The
microfluidic format used in conjunction with soft materials was demonstrated to yield
1060 min total assay times, a 103-fold reduction of antibody and buffer consumption, 5 pM
detection limits, and a 3.6 log dynamic range of detection.
FIG. 19. Patterning two different antibody layers on the same silicon surface: (1) immobilization of mouse IgG with
LMPA protection layer, (2) photoresist patterning, (3) oxygen plasma etching followed by photoresist strip, and (4) immo-
bilization of human IgG and removal of protective LMPA pattern using GELase. Adapted with permission from W.-C.
Sung et al., Anal. Chem. 81, 7967 (2009). Copyright 2009 American Chemical Society.
also tested with 70% of enzyme activity maintained. Sung et al. reported photopatterned 3-D
hydrogel plugs for antibody immobilization on a PDMS surface.97 In order to anchor PA gel to
the PDMS (which lacks functional groups), the PDMS was first treated with oxygen plasma.
Then, PEI and PAA solutions were injected to yield a multilayer coating on the PDMS surface.
Subsequently, an EDC and NHS chemistry was used to covalently bind the PEI and PAA layers
together. The photoinitiator benzophenone was injected and diffused through the PDMS surface.
Protein G was acrylate-functionalized using NHS-PEG-acrylate. A hydrogel precursor consisting
of acrylamide/bis-acrylamide and acrylated-PEG-protein G was introduced. Upon exposure to
UV light using a 40 objective lens, a cylindrical hydrogel plug was formed in the microfluidic
channel via free-radical polymerization initiated from the surface-buried benzophenone. Mouse
IgG was immobilized to the protein G and detected using HRP conjugated anti-mouse IgG.
Also, ERc (estrogen receptor c) was detected in the same format, showing a sensitivity 13 times
higher than with ERc adsorbed on the bare PDMS surface.
B. Photoactivated detachment
Light-activated spatial control of proteins is not limited to site-specific patterning, but ame-
nable to site-specific de-patterning (i.e., photocleaving), useful for on-demand protein elution.
Kims group reported on aptamer-based microfluidic affinity chromatography using a photo-
cleavable linker for concentration and elution of proteins.62,88 First, magnetic beads were
prepared by coating a nanomagnetite core with PS and divinylbenzene polymer. Amine-
functionalized PEG, copolymerized in the bead shell, helped ensure bead solubility, reduced
nonspecific protein binding, and allowed aptamer crosslinking. An in-house synthesized
photocleavable linker (4-(4-(1-hydroxyethyl)-2-methoxy-5-nitrophenoxy butanoic acid) was im-
mobilized on the amino functionalized beads using NHS and carbodiimide (1,3-diisopropylcar-
bodiimide, DIC) chemistry. Aminated aptamer for HCV (hepatitis C virus) RNA polymerase
was immobilized via an NHS functional group of the photocleavable linker. After magnetic
beads were captured in a central chamber of a microfluidic device using a permanent magnet,
HCV RNA polymerase in patient serum was captured, concentrated, and eluted upon UV expo-
sure. Eluted enzyme was digested off-chip by trypsin and analyzed by MALDI-TOF/MS with
an estimated detection limit of 9.6 fmol. The authors observed improved assay performance, as
compared to antibody-based capture, because site-specific immobilization of aptamers through
amination at the 30 - and 50 -positions maximizes antigen binding activity. Further, aptamers were
not appreciably degraded by protease in serum. The linker showed 70% cleaving activity. In
follow-up work, a similar photoelution method was used to analyze FITC-labeled proteins.88 In
041501-36 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
a microfluidic chip made by anodic boding of glass and silicon, magnetic beads functionalized
with a photocleavable linker [4-(4-(1-(9-fluorenylmethoxycarbonylamino)ethyl)-2-methoxy-5-
nitrophenoxy)-butanoic acid] were pseudo-immobilized via microposts. Two types of photo-
cleavable linkers were prepared; one with biotin and one with NHS ester (Figure 20(a)). Biotin
was conjugated to the bead with the BOP [benzotriazol-1-yloxytris(dimethylamino)phospho-
nium hexafluorophosphate] chemistry. Aminated aptamer for HCV RNA replicase was attached
to the magnetic beads using NHS and DIC chemistry. Biotin-conjugated beads were used for
purification of streptavidin and aptamer conjugated beads were used for purification of the
HCV RNA replicase. Target proteins were mixed with model proteins (HSA, ovalbumin),
injected into the packed bed, concentrated, and then photoeluted upon UV exposure (Figure
20(b)). The fluorescence signal was monitored for detection.
FIG. 20. (a) Structures of the photo-cleavable sites on the bead. (1) Biotinylated bead with a short spacer and (2) active
ester containing bead with a long spacer for aptamer coupling. (b) Schematic view of microaffinity purification process. (1)
Injection of the protein mixture into the microchip packed with microbeads, (2) purification of the target protein, (3) UV
irradiation, and (4) analysis of the photolytically eluted protein. Reprinted with permission from W. J. Chung et al.,
Electrophoresis 26, 694 (2005). Copyright 2005 Wiley InterScience.
041501-37 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
FIG. 21. Schematic representations of the temperature-responsive bead immunoassay system. (a) A 100 nm diameter latex
nanobead is surface-conjugated with biotinPEG and PNIPAM. Streptavidin is bound to the exposed biotin, providing
binding sites for the biotinylated anti-digoxin IgG, and (b) a schematic of the experimental protocol. (1) Suspended beads
are loaded into the microfluidic channel, (2) the temperature in the channel is then increased from room temperature to
37 C, resulting in aggregation and adhesion of the beads to the channel wall, (3) flow is initiated, washing unadsorbed
beads out of the channel, (4) a mixture of fluorescently labeled digoxigenin and digoxin is flowed into the channel, (5) com-
ponents of this mixture that fail to bind the immobilized antibodies are washed through, and (6) finally, the temperature in
the channel is reduced, and the aggregation-absorption process is reversed as antigen-bound beads leave the channel with
the flow stream. Reprinted with permission from N. Malmstadt et al., Lab Chip 4, 412 (2004). Copyright 2004 The Royal
Society of Chemistry.
FIG. 22. (a) Electrochemically activated protein immobilization within a sealed microchannel. (1) Introduce a blocking
agent (polyethyleneimine (PEI) and heparin) through the microchannel for antibiofouling, (2) generate HBrO local to the
microelectrode, which removes a portion of blocking agent, thus making this part of the channel bottom available to protein
physisorption, and (3) introduce proteins into the microchannel for immobilization. Reprinted with permission from H.
Kaji et al., Anal. Chem. 78, 5469 (2006). Copyright 2006 American Chemical Society. (b) Chitosan-based electrochemi-
cally activated protein immobilization on gold electrode. (1) pH dependent protonation/deprotonation of the chitosan mole-
cule, (2) schematic view of chitosan deposition, and (3) schematic view of chitosan deposition in a microfluidic channel.
Reprinted with permission from J. J. Park et al., Lab Chip 6, 1315 (2006). Copyright 2006 The Royal Society of
Chemistry.
pH.130,185 Thus, when the pH was increased by hydrolysis (OH generation) on the electrode
surface, chitosan was assembled into a hydrogel network. The microfluidic device was made by
clamping a glass wafer housing SU-8 microchannels to a PDMS-coated Plexiglas wafer. Gold
electrodes were patterned on the glass wafer. After the chitosan solution was injected, the gold
electrode was negatively biased for 240 s. Negatively charged gold electrodes electrostatically
attracted positively charged chitosan, and a local alkaline pH turned chitosan insoluble, thus
depositing the species on the electrode surface. Amine groups, rich on chitosan, were activated
with GA, and then protein was immobilized in a site-specific manner. GFP was immobilized
and the fluorescence image indicated that GFP was active in this chitosan hydrogel environ-
ment. In follow-up work, an enzyme was immobilized on an in situ assembled chitosan hydro-
gel in a reversible manner.186 First, pro (tyrosine) tag was genetically fused to the enzyme Pfs
(S-adenosylhomocysteine nucleosidase). In the presence of tyrosinase and pro-tagged Pfs, Pfs
was covalently bound to chitosan (i.e., through generation of reactive O-quinone). Covalent
bonds were further stabilized by NaBH3CN. Upon biasing, Pfs-chitosan was deposited onto the
gold electrode to form a hydrogel. The enzymatic activity of Pfs was characterized, and the ac-
tivity in the chitosan hydrogel was higher than that of enzyme in solution. The chitosan assem-
bly disintegrated with a mild acid treatment allowing renewal of the electrode surface.
from this earlier work of DNA microarrays.187,206,207 Thus, adaptation of DNA-directed protein
immobilization for multiplexed microfluidic protein assays is attractive. A prerequisite for such
immobilization is patterning of capture ssDNA of a specific sequence on a predetermined loca-
tion of surface. Patterning is often done using a spot printer.25,26 Possible drawbacks for spot
printing are the high cost and the need to print before a chip is assembled, which may denature
proteins and limit chip substrate material depending on the chip finishing processes needed.
Schroeder et al. reported on microfluidic reaction vessels for DNA-directed multiplexed
immunoassays (Figure 23).25 The PMMA chip consists of 12 wells, in which 4 4 microarrays
for capture cDNAs are housed. Each well has microfluidic access for reagent injection and
washing. First, a carboxylic acid group of the photolinker 4-benzoylbenzoic acid was activated
with carbodiimide (1,3-dicyclohexylcarbodiimide) and NHS. Then, cDNAs were crosslinked to
the PMMA surface using UV exposure via hydrogen abstraction of benzophenone from C-H
bonds on the PMMA surface.75 The four cDNAs were spot-patterned in the chip wells. Using
the activated photolinker, four different amino-modified cDNA oligonucleotides were function-
alized with benzophenone. Second, streptavidin was covalently attached to DNAs complemen-
tary to the surface-immobilized cDNAs. Streptavidin was conjugated with sulfo-SMPB, which
then covalently linked to the thiolated DNA. Four different biotinylated antibodies to protein
targets PSA, TNF-a, interleukin 6 (IL6), and interleukin 23 (IL23) were attached to each
streptavidin-functionalized DNA molecule via bioaffinity interaction. The antibody mixture was
conjugated with DNA and injected into the chip wells to selectively immobilize each antibody
on a specific cDNA spot. Then, a multiplexed sandwich immunoassay was demonstrated. Gold-
nanoparticle conjugated antibodies were used for light-transmission detection via catalytic silver
deposition.
Extension of DNA-directed protein immobilization was conducted to realize the DEAL
(DNA-encoded antibody libraries) platform by Heaths group. DEAL simultaneously detects
ssDNA and proteins and can sort cells for multiparameter analysis.26 The rationale for DNA
immobilization on the DEAL platform stems from the realization that surface chemistry for
immobilization of different classes of biomolecules may not be compatible. Thus, by using
DNA-mediated immobilization, such incompatibility issues could be alleviated. Antibodies are
conjugated with SANH (succinimidyl 4-hydrazinonicotinate acetone hydrazine) in order to
introduce hydrazide groups. Aminated DNA was aldehyde-functionalized using AFB
FIG. 23. Schematic drawing of multiplexed immunoassay performed in the wells of a disposable microarray. The different
sandwich assays were assembled by site-specific DNA-directed immobilization to the dedicated capture probes cD-cG,
illustrated in the scheme. Reprinted with permission from H. Schroeder et al., Anal. Chem. 81, 1275 (2009). Copyright
2009 American Chemical Society.
041501-40 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
F. Reversible immobilization
Covalent bonds are usually irreversible, meaning that microfluidic chips relying on covalent
immobilization strategies are typically used once and then discarded. Alternatively, the immobi-
lization surface can be refreshed using a strong oxidizer.46 Bioaffinity interactions like DNA
hybridization or protein A/G binding to antibody are reversible using strong acids/bases, con-
centrated salts, or heat treatments. Physisorption, especially electrostatic interactions, can be
reversed by increasing salt concentrations.20 The cost of the assay can be reduced and multiple
assays can be performed in a single device if the surface can be regenerated reproducibly.
Li et al. used a chelating agent to refresh an enzyme-immobilized surface on magnetic
beads.63 The concept employed is that trypsin can be immobilized via a Lewis acid-base interac-
tion between metal ions and an electron acceptor group on the protein surface (e.g., imidazole of
histidine).208,209 For elution, a competitive reaction with EDTA can break the bond to release
the enzyme. First, metal-ion chelated magnetic beads were prepared. Magnetic cores were syn-
thesized using a solvothermal reaction of FeCl36H2O in ethylene glycol. Then, the magnetic
core was coated with silica via sol-gel formation of tetraethyl orthosilicate (TEOS). Second, imi-
nodiacetic acid (IDA), a metal chelating agent, was covalently attached to the silanizing agent 3-
glycidoxypropyltrimethoxysilane agent GLYMO (i.e., GOPTS) via reaction of the glycidyl group
with secondary amines. GLYMO-IDA was then attached to the silica shell of the magnetic beads
by a silane-coupling reaction. Copper ions (Cu2) and TPCK (tosyl phenylalanyl chloromethyl
ketone)-treated trypsin were incubated with the magnetic beads. The enzyme was bound to the
bead via the Lewis acid-base interaction with the divalent cation chelator, IDA (Figure 24). The
magnetic beads were captured in a microfluidic channel by a permanent magnet. Cytochrome c
and BSA were digested by trypsin, and analyzed with MALDI-TOF/MS. The magnetic beads
were readily packed, flushed, and repacked by magnetic-field and fluidic control. Compared to co-
valently bound enzymes, IDA-metal chelated adsorption was regenerated easily by adding compet-
ing EDTA solution to remove metal ions. Regenerated enzyme showed reproducible results up to
4 runs. Bead-based solid-phase digestion was faster than in-solution trypsin digestion.
FIG. 24. Process of Cu-IDA-GLYMO-MS microspheres preparation and trypsin immobilization. Reprinted with permis-
sion from Y. Li et al., J. Proteome Res. 6, 2367 (2007). Copyright 2007 American Chemical Society.
was tested via digestion of ArgOEt (arginine ethyl ester) to Arg, and electrophoretic separation
of the substrate and product. Note that the process required almost two days to completely im-
mobilize trypsin to the PMBN polymer. Jons group prepared amphiphilic polymers with hydro-
phobic groups anchoring to COC surfaces, PEG protein-repelling group, and NHS-ester groups
for covalent protein immobilization (Figure 25).210 The multifunctional polymers poly(DMA-r-
PEGMA-r-NAS) and poly(BMA-r-PEGMA-r-NAS) were prepared; hydrophobic dodecyl meth-
acrylate (DMA) or hydrophobic benzyl methacrylate (BMA), hydrophilic polyethylene glycol
methacrylate (PEGMA), and N-acryloylsuccinimide (NAS, a reactive NHS ester of acrylic
monomer) were radical-copolymerized using AIBN. Incubation of an injection-molded COC de-
vice in the polymer solution for 1 h completed surface coating. The surface anchoring properties,
protein repelling functionality, and covalent immobilization of antibodies were tested. Poly(BMA-
r-PEGMA-r-NAS) showed better antibiofouling and higher antibody immobilization capacity.
Therefore, sandwich immunoassays of troponin I were demonstrated on poly(BMA-r-PEGMA-r-
NAS) surfaces, resulting in a 10 ng/ml detection limit. In another study, the authors also used a
similar multifunctional surface to immobilize proteins on a PS surface.211 A major difference from
Ref. 210 was that an additional step for activating methacrylate with EDC and NHS was used
before protein immobilization. Amine-functionalized biotin was covalently linked to the activated
surface to attach biotinylated antibodies via streptavidin-biotin linkage. In addition, protein A was
covalently linked to the surface to attach multiple antibodies for immunoassays.
FIG. 25. (a) Chemical structures of the multifunctional amphiphilic polymers, and (b) schematic representation of the pro-
cedure for immobilizing biomolecules onto a polymer-modified COC surface with antibiofouling properties. Reprinted
with permission from D. Sung et al., Biosens. Bioelectron. 26, 3967 (2011). Copyright 2011 Elsevier.
protein immobilization are being generated at an amazing pace. The basis for most strategies
originates in three general immobilization mechanisms (physisorption, bioaffinity interaction,
and covalent bonding), and a combination of the three mechanisms. A key aim of immobiliza-
tion strategies is improving assay sensitivity, specificity, reproducibility, and sometimes
throughput. To meet this goal, researchers are focused on (1) forming stable and strong bonds
between protein and immobilization surface, (2) minimizing nonspecific adsorption of bioma-
cromolecules or cells, (3) keeping the protein in an active state, and (4) orienting protein for
unhindered access of binding partners. In some instances, the use of physisorption, bioaffinity
interaction, or covalent bonding alone does not render meeting this goal possible.
Consequently, various combinations of these immobilization mechanisms are frequently used
with important outcomes.
Regardless of a weak and irreproducible nature, physisorption is often employed in protein
immobilization, especially when quickly proving a new assay concept. Nevertheless, physisorption,
especially electrostatic interaction, can be strong enough that immobilized proteins remain attached
even in the presence of strong external forces, such as the hydrodynamic shear force, magnetic
forces, or electrostatic forces. In some cases, physisorption is achieved almost instantly, compared
to covalent interactions usually requiring long incubation periods. Thus, multistep assays such as
Western blotting are possible using the physisorption (e.g., electrostatic protein blotting right after
electrophoretic separation without degradation of protein separation performance).
Bioaffinity interactions are extensively used in literature owing to its relatively strong, high
specific, oriented protein immobilization, and in some instances, the possibility of reversing
linkage. However, we noted that bioaffinity interaction was generally used in conjunction with
covalent linkage chemistries and/or physisorption. The streptavidin-biotin interaction is widely
used in bioanalytical chemistry, owing to exceptional binding strength, specificity, and multiple
binding sites for increased binding capacity. Protein A and protein G are also popular binding
agents, especially in immunoassays, because both bind to the Fc region of IgG and do not block
the Fab region, consequently improving the antigen-binding capability of immobilized IgG.
Another benefit is that protein A and G binding can be reversed using suitable chemical
treatments.
041501-43 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
DNA hybridization is a promising bioaffinity strategy for immobilization, especially for multi-
plexed or multiparameter assays. DNA-encoded immobilization relies on a specific hybridization
between a single-stranded DNA (ssDNA) sequence and its complementary sequence. DNA-
directed immobilization originates from DNA microarray technology, which offers an enormous
multiplexing capability. After an ssDNA-encoded protein is attached to its complementary DNA, a
protein microarray is formed in a microfluidic device for a multiplexed assay. Although analysis
of only a handful of species has been demonstrated, a strong potential exists for this bioaffinity-
based multiplexed immobilization scheme. Numerous efforts have been undertaken to replace anti-
body with aptamers for capture of antigen. The benefits of aptamers are potentially numerous com-
pared to those of antibodies and include: smaller size for less steric hindrance, no need for animals
in production, lower costs to produce, longer shelf life, and less sensitivity to environmental condi-
tions. Nevertheless, antibodies are still the dominant capture reagent.
Covalent bonds are the strongest and most frequently employed protein immobilization
mechanism. Amine-based covalent linking is widely employed for protein immobilization
owing to rich lysine residues on protein surfaces. Glutaraldehyde activation of amine is popular
technique in microfluidics, followed by NHS ester and carbodiimide. A downside of amine-
based chemistry is that multiple bonds can be formed, since numerous lysine residues exist on
a protein surface, which may block active sites of the protein or cause conformational change.
Thus, more specific, simple, and protein-friendly covalent linking methods would fill an exist-
ing gap. Covalent linkage is often used with a spacer molecule like PEG or hydrophilic poly-
mer coatings to reduce steric hindrance and the possibility of protein denaturation.
Most microfluidic protein assays find immobilization of analyte on the inner surface of a
microfluidic channel, which is rational given that the core of the microchannels are used for
transport of reagent, analytes, substrate, and sample. Immobilization methods depend on the
immobilization surface. More and more efforts are devoted to identifying strategies for immobi-
lizing proteins on plastic surfaces, as plastics are becoming widely used in microfluidic devices
owing to the low cost and mass-production potential. PDMS remains a popular substrate for
rapid prototyping of new devices. Thus, new immobilization methods for PDMS surfaces have
been an active area of research. Owing to the inert surfaces (i.e., lacking functional groups),
immobilization chemistries for plastic and PDMS are not straightforward. In contrast to plastic
and PDMS, the formation of silanol groups is a routine starting point of the immobilization pro-
cess on glass or silicon surfaces. Oxygen-plasma treatment or less frequently solution-phase ox-
idation are used to prepare plastics and PDMS, but as evidenced by many studies, the linkages
formed on the oxidized polymer surface are weak and unstable. Consequently, a surface-grafted
polymer or physisorbed functional layer on the channel surface is a major immobilization strat-
egy being pursued for plastics or PDMS substrates. Exploiting the hydrophobicity of PDMS or
plastics is a straightforward way to immobilize proteins having hydrophobic patches on the bio-
molecular surface. Although hydrophobic interaction is still used often in benchtop biochemical
assays such as Western blotting and dot blots, hydrophobic interactions are not generally rec-
ommended if tight reproducibility and quantitative assays are needed.
Various surface coatings including inorganic (e.g., sol-gel, gold/silver nanoparticle), poly-
meric (e.g., PEI, PAA, PDADMAC, PVA, PEG gel, and polyacrylamide gel), and biopolymeric
(dextran, chitosan, and protein aggregates) coatings, help to minimize nonspecific adsorption
and improve the activity of immobilized proteins. A recent trend has been to incorporate multi-
ple functions (e.g., activated functional group for protein immobilization, antibiofouling layer
for reduced nonspecific protein adsorption, and surface anchoring group) in a single copolymer,
to obviate the time-consuming and error-prone layering of multiple functional coatings. Thus,
devising effective and smart coating layers are emerging as a major research topic.
Three-dimensional solid supports like hydrogels, packed beads, sol-gels, and polymer
monoliths yield high surface-area-to-volume ratios that underpin efficient material capture,
improved binding kinetics owing to short diffusion lengths in the materials, and in some cases
provide water-retaining protein-friendly environments (e.g., hydrogels). Devising immobiliza-
tion chemistries that are compatible with these solid supports will continue to be a worthy
research topic.
041501-44 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
Researchers are adding more and more functionalities to microfluidic devices to meet the
ambitious goal of microfluidics: a truly integrated, samples-in-answers-out stand-alone
platform. Various smart immobilization techniques have been introduced. One prominent
smart immobilization method is light-directed immobilization. Multiple proteins can be pat-
terned with excellent spatial control (depending on the exposure system used) after a microflui-
dic chip is completely sealed. Microcontact printing, spot printing, and other soft lithography
techniques are limited to analyte patterning before assembly of the chip. Early literature
employed reversible sealing of PDMS on glass in order to perform multiplexing-oriented pat-
terning methods (e.g., the lFN platform by Delamarches group). However, leakage or cross-
talk between microchannels could be a challenge of this approach. An advantage of light-
activated immobilization over electrochemistry-mediated protein patterning is that electrodes do
not need to be patterned inside the microfluidic chip, which simplifies design and fabrication of
the microfluidic device. Light-activated immobilization is facile, compared to covalent chemis-
tries in which a long incubation period is required. Thus, light-activated immobilization was
used in microfluidic Western blotting in which protein should be immobilized as soon as elec-
trophoretic separation is completed in order to maintain high separation resolution. A downside
could be that the light-activated immobilization may require expensive optics and motorized
stages to precisely pattern protein patches in such small microfluidic channels. Light has also
been successfully employed to cleave a bond to release biomolecules from the surface. A ther-
mal transition between hydrophobic to hydrophilic surfaces may provide a quick and easy way
to capture and release proteins. Heat may not need to be transferred to the temperature respon-
sive polymer using microfabricated heaters. Instead, infrared radiation or injection of hot fluid
could also be used to activate temperature-responsive immobilization surfaces, thus simplifying
device fabrication and assay operation. Reversible immobilization has been realized by simply
introducing chelating agents like EDTA, allows the immobilized protein to be eluted from an
immobilization surface. Even though the reproducibility of protein capture efficiency on a
rejuvenated surface is limited, continued studies may be worthwhile because covalently
attached proteins can only be removed by completely renewing the immobilization surface
(e.g., using a strong oxidizing agent).
Many smart immobilization strategies could be combined for more sophisticated multi-
analyte assays required for complex sample analyses. For example, light, electrochemical, or
thermal stimuli are orthogonal, meaning that they can be used simultaneously to address a
specific site at a certain time point without affecting other responsive functions. Ultimately, for
a highly integrated multiparameter assay, biomolecules such as proteins and oligonucleotides as
well as cells could be injected into the microfluidic device, immobilized on different spots, and
released on demand using these mutually independent stimuli. Clearly, protein immobilization
strategies play a critical role in the measurement of biological systems and processes. We look
forward to continued substantial innovation in this area.
1
V. Gauci, E. Wright, and J. Coorssen, J. Chem. Biol. 4, 3 (2011).
2
E. F. Petricoin, K. C. Zoon, E. C. Kohn, J. C. Barrett, and L. A. Liotta, Nat. Rev. Drug Discovery 1, 683 (2002).
3
C. A. Borrebaeck, Immunol. Today 21, 379 (2000).
4
J. A. Ludwig and J. N. Weinstein, Nat. Rev. Cancer 5, 845 (2005).
5
K. M. Koeller and C.-H. Wong, Nature 409, 232 (2001).
6
J. B. van Beilen and Z. Li, Curr. Opin. Biotechnol. 13, 338 (2002).
7
J. P. Rasor and E. Voss, Appl. Catal. A 221, 145 (2001).
8
A. H. Ng, U. Uddayasankar, and A. R. Wheeler, Anal. Bioanal. Chem. 397, 991 (2010).
9
T. G. Henares, F. Mizutani, and H. Hisamoto, Anal. Chim. Acta 611, 17 (2008).
10
A. Bange, H. B. Halsall, and W. R. Heineman, Biosens. Bioelectron. 20, 2488 (2005).
11
J. Krenkova and F. Foret, Electrophoresis 25, 3550 (2004).
12
Y. Asanomi, H. Yamaguchi, M. Miyazaki, and H. Maeda, Molecules 16, 6041 (2011).
13
M. Miyazaki and H. Maeda, Trends Biotechnol. 24, 463 (2006).
14
G. M. Whitesides, Nature 442, 368 (2006).
15
M. A. Burns, B. N. Johnson, S. N. Brahmasandra, K. Handique, J. R. Webster, M. Krishnan, T. S. Sammarco, P. M.
Man, D. Jones, D. Heldsinger, C. H. Mastrangelo, and D. T. Burke, Science 282, 484 (1998).
16
C. J. Easley, J. M. Karlinsey, J. M. Bienvenue, L. A. Legendre, M. G. Roper, S. H. Feldman, M. A. Hughes, E. L.
Hewlett, T. J. Merkel, and J. P. Ferrance, Proc. Natl. Acad. Sci. U.S.A. 103, 19272 (2006).
17
P. Liu, X. Li, S. A. Greenspoon, J. R. Scherer, and R. A. Mathies, Lab Chip 11, 1041 (2011).
041501-45 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
18
M. He and A. E. Herr, J. Am. Chem. Soc. 132, 2512 (2010).
19
S. Q. Tia, M. He, D. Kim, and A. E. Herr, Anal. Chem. 83, 3581 (2011).
20
D. Kim, K. Karns, S. Q. Tia, M. He, and A. E. Herr, Anal. Chem. 84, 2533 (2012).
21
A. J. Hughes, R. K. Lin, D. M. Peehl, and A. E. Herr, Proc. Natl. Acad. Sci. U.S.A. 109, 5972 (2012).
22
A. J. Hughes and A. E. Herr, Proc. Natl. Acad. Sci. U.S.A. 109, 21450 (2012).
23
M. He, J. Novak, B. A. Julian, and A. E. Herr, J. Am. Chem. Soc. 133, 19610 (2011).
24
M. Chung, D. Kim, and A. E. Herr, Microchamber Western blotting using poly-L-lysine conjugated polyacrylamide gel
for blotting of SDS coated proteins, Anal. Chem. (to be published).
25
H. Schroeder, M. Adler, K. Gerigk, B. Muuller-Chorus, F. Go utz, and C. M. Niemeyer, Anal. Chem. 81, 1275 (2009).
26
R. C. Bailey, G. A. Kwong, C. G. Radu, O. N. Witte, and J. R. Heath, J. Am. Chem. Soc. 129, 1959 (2007).
27
M. Wolf, D. Juncker, B. Michel, P. Hunziker, and E. Delamarche, Biosens. Bioelectron. 19, 1193 (2004).
28
A. Bhattacharyya and C. Klapperich, Biomed. Microdevices 9, 245 (2007).
29
C. J
onsson, M. Aronsson, G. Rundstr om, C. Pettersson, I. Mendel-Hartvig, J. Bakker, E. Martinsson, B. Liedberg, B.
MacCraith, O. Ohman, and J. Melin, Lab Chip 8, 1191 (2008).
30
Y.-N. Yang, H.-I. Lin, J.-H. Wang, S.-C. Shiesh, and G.-B. Lee, Biosens. Bioelectron. 24, 3091 (2009).
31
K.-S. Shin, S. W. Lee, K.-C. Han, S. K. Kim, E. K. Yang, J. H. Park, B.-K. Ju, J. Y. Kang, and T. S. Kim, Biosens.
Bioelectron. 22, 2261 (2007).
32
N. Malmstadt, A. S. Hoffman, and P. S. Stayton, Lab Chip 4, 412 (2004).
33
D. Yang, X. Niu, Y. Liu, Y. Wang, X. Gu, L. Song, R. Zhao, L. Ma, Y. Shao, and X. Jiang, Adv. Mater. 20, 4770
(2008).
34
Q. Xiang, G. Hu, Y. Gao, and D. Li, Biosens. Bioelectron. 21, 2006 (2006).
35
H. Wang, Y. Liu, C. Liu, J. Huang, P. Yang, and B. Liu, Electrochem. Commun. 12, 258 (2010).
36
S. N. Tan, L. Ge, H. Y. Tan, W. K. Loke, J. Gao, and W. Wang, Anal. Chem. 84, 10071 (2012).
37
K. Schult, A. Katerkamp, D. Trau, F. Grawe, K. Cammann, and M. Meusel, Anal. Chem. 71, 5430 (1999).
38
Y. Dong, K. Phillips, and Q. Cheng, Lab Chip 6, 675 (2006).
39
T. Yang, S.-Y. Jung, H. Mao, and P. S. Cremer, Anal. Chem. 73, 165 (2001).
40
J. Yakovleva, R. Davidsson, A. Lobanova, M. Bengtsson, S. Eremin, T. Laurell, and J. Emneus, Anal. Chem. 74, 2994
(2002).
41
B. Cannon, N. Weaver, Q. Pu, V. Thiagarajan, S. Liu, J. Huang, M. W. Vaughn, and K. H. Cheng, Langmuir 21, 9666
(2005).
42
E. T. Castellana, S. Kataoka, F. Albertorio, and P. S. Cremer, Anal. Chem. 78, 107 (2006).
43
C. Hou and A. E. Herr, Anal. Chem. 82, 3343 (2010).
44
A. E. Herr, A. V. Hatch, D. J. Throckmorton, H. M. Tran, J. S. Brennan, W. V. Giannobile, and A. K. Singh, Proc. Natl.
Acad. Sci. U.S.A. 104, 5268 (2007).
45
K. Karns and A. E. Herr, Anal. Chem. 83, 8115 (2011).
46
M. He and A. E. Herr, Nat. Protoc. 5, 1844 (2010).
47
D. A. Markov, K. Swinney, and D. J. Bornhop, J. Am. Chem. Soc. 126, 16659 (2004).
48
D. Gerber, S. J. Maerkl, and S. R. Quake, Nat. Methods 6, 71 (2009).
49
E. Ouellet, C. Lausted, T. Lin, C. W. T. Yang, L. Hood, and E. T. Lagally, Lab Chip 10, 581 (2010).
50
X. Chen, M. A. Kapil, A. J. Hughes, and A. E. Herr, Anal. Chem. 83, 6573 (2011).
51
S. Q. Tia, K. Brown, D. Chen, and A. E. Herr, Anal. Chem. 85, 2882 (2013).
52
M. S. Thomsen, P. P olt, and B. Nidetzky, Chem. Commun. 2007, 2527.
53
A. J. Hughes and A. E. Herr, Anal. Chem. 82, 3803 (2010).
54
M. Baeza, C. L opez, J. Alonso, J. L
opez-Santn, and G. Alvaro, Anal. Chem. 82, 1006 (2010).
55
H. Mao, T. Yang, and P. S. Cremer, Anal. Chem. 74, 379 (2002).
56
P. He, G. Greenway, and S. J. Haswell, Microfluid. Nanofluid. 8, 565 (2010).
57
W.-G. Koh and M. Pishko, Sens. Actuators B 106, 335 (2005).
58
S. B. Brueggemeier, S. J. Kron, and S. P. Palecek, Anal. Biochem. 329, 180 (2004).
59
S. Kostler, V. Ribitsch, K. Stana-Kleinschek, G. Jakopic, and S. Strnad, Colloids Surf., A 270271, 107 (2005).
60
S. Ekstr
om, P. Onnerfjord, J. Nilsson, M. Bengtsson, T. Laurell, and G. Marko-Varga, Anal. Chem. 72, 286 (2000).
61
D. S. Peterson, T. Rohr, F. Svec, and J. M. Frechet, Anal. Chem. 74, 4081 (2002).
62
S. Cho, S. H. Lee, W. J. Chung, Y. K. Kim, Y. S. Lee, and B. G. Kim, Electrophoresis 25, 3730 (2004).
63
Y. Li, X. Xu, B. Yan, C. Deng, W. Yu, P. Yang, and X. Zhang, J. Proteome Res. 6, 2367 (2007).
64
H. Wu, Y. Tian, B. Liu, H. Lu, X. Wang, J. Zhai, H. Jin, P. Yang, Y. Xu, and H. Wang, J. Proteome Res. 3, 1201 (2004).
65
K. Jo, M. L. Heien, L. B. Thompson, M. Zhong, R. G. Nuzzo, and J. V. Sweedler, Lab Chip 7, 1454 (2007).
66
S. S. Park, S. I. Cho, M. S. Kim, Y. K. Kim, and B. G. Kim, Electrophoresis 24, 200 (2003).
67
J. Gao, J. Xu, L. E. Locascio, and C. S. Lee, Anal. Chem. 73, 2648 (2001).
68
J. Yakovleva, R. Davidsson, M. Bengtsson, T. Laurell, and J. Emneus, Biosens. Bioelectron. 19, 21 (2003).
69
Y. S. Huh, A. J. Chung, and D. Erickson, Microfluid. Nanofluid. 6, 285 (2009).
70
J.-W. Choi, K. W. Oh, J. H. Thomas, W. R. Heineman, H. B. Halsall, J. H. Nevin, A. J. Helmicki, H. T. Henderson, and
C. H. Ahn, Lab Chip 2, 27 (2002).
71
J. R. Anderson, D. T. Chiu, H. Wu, O. J. Schueller, and G. M. Whitesides, Electrophoresis 21, 27 (2000).
72
J. M. K. Ng, I. Gitlin, A. D. Stroock, and G. M. Whitesides, Electrophoresis 23, 3461 (2002).
73
S. K. Sia and G. M. Whitesides, Electrophoresis 24, 3563 (2003).
74
H. Becker and L. E. Locascio, Talanta 56, 267 (2002).
75
D. M. Dankbar and G. Gauglitz, Anal. Bioanal. Chem. 386, 1967 (2006).
76
H. Becker and C. Gartner, Electrophoresis 21, 12 (2000).
77
S. Laib and B. D. MacCraith, Anal. Chem. 79, 6264 (2007).
78
Y. Bai, C. G. Koh, M. Boreman, Y.-J. Juang, I.-C. Tang, L. J. Lee, and S.-T. Yang, Langmuir 22, 9458 (2006).
79
Y. Yuan, H. He, and L. J. Lee, Biotechnol. Bioeng. 102, 891 (2009).
80
X. Wen, H. He, and L. J. Lee, J. Immunol. Methods 350, 97 (2009).
041501-46 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
81
A. Y. Rubina, A. Kolchinsky, A. A. Makarov, and A. S. Zasedatelev, Proteomics 8, 817 (2008).
82
A. G. Lee, C. P. Arena, D. J. Beebe, and S. P. Palecek, Biomacromolecules 11, 3316 (2010).
83
A. G. Lee, D. J. Beebe, and S. P. Palecek, Biomed. Microdevices 14, 247 (2012).
84
J. De Jong, R. Lammertink, and M. Wessling, Lab Chip 6, 1125 (2006).
85
D. S. Peterson, Lab Chip 5, 132 (2005).
86
G. H. Seong and R. M. Crooks, J. Am. Chem. Soc. 124, 13360 (2002).
87
Y.-C. Wang and J. Han, Lab Chip 8, 392 (2008).
88
W. J. Chung, M. S. Kim, S. Cho, S. S. Park, J. H. Kim, Y. K. Kim, B. G. Kim, and Y. S. Lee, Electrophoresis 26, 694 (2005).
89
K. Sato, M. Yamanaka, H. Takahashi, M. Tokeshi, H. Kimura, and T. Kitamori, Electrophoresis 23, 734 (2002).
90
Y. H. Tennico, D. Hutanu, M. T. Koesdjojo, C. M. Bartel, and V. T. Remcho, Anal. Chem. 82, 5591 (2010).
91
T. Richter, L. L. Shultz-Lockyear, R. D. Oleschuk, U. Bilitewski, and D. J. Harrison, Sens. Actuators B 81, 369 (2002).
92
V. Sivagnanam, B. Song, C. Vandevyver, and M. A. Gijs, Anal. Chem. 81, 6509 (2009).
93
K. Sato, M. Tokeshi, T. Odake, H. Kimura, T. Ooi, M. Nakao, and T. Kitamori, Anal. Chem. 72, 1144 (2000).
94
M. He and A. E. Herr, Anal. Chem. 81, 8177 (2009).
95
W. Zhan, G. H. Seong, and R. M. Crooks, Anal. Chem. 74, 4647 (2002).
96
K. H. Park, H. G. Park, J.-H. Kim, and K. H. Seong, Biosens. Bioelectron. 22, 613 (2006).
97
W.-C. Sung, H.-H. Chen, H. Makamba, and S.-H. Chen, Anal. Chem. 81, 7967 (2009).
98
K. Sakai-Kato, M. Kato, and T. Toyooka, Anal. Chem. 75, 388 (2003).
99
J. Ji, Y. Zhang, X. Zhou, J. Kong, Y. Tang, and B. Liu, Anal. Chem. 80, 2457 (2008).
100
H. Hisamoto, Y. Shimizu, K. Uchiyama, M. Tokeshi, Y. Kikutani, A. Hibara, and T. Kitamori, Anal. Chem. 75, 350 (2003).
101
R. P. Sebra, K. S. Masters, C. Y. Cheung, C. N. Bowman, and K. S. Anseth, Anal. Chem. 78, 3144 (2006).
102
S. Zhang, Nature Mater. 3, 7 (2004).
103
A. W. Martinez, S. T. Phillips, G. M. Whitesides, and E. Carrilho, Anal. Chem. 82, 3 (2010).
104
C. M. Cheng, A. W. Martinez, J. Gong, C. R. Mace, S. T. Phillips, E. Carrilho, K. A. Mirica, and G. M. Whitesides,
Angew. Chem., Int. Ed. 49, 4771 (2010).
105
J. L. Osborn, B. Lutz, E. Fu, P. Kauffman, D. Y. Stevens, and P. Yager, Lab Chip 10, 2659 (2010).
106
F. Rusmini, Z. Zhong, and J. Feijen, Biomacromolecules 8, 1775 (2007).
107
K. Nakanishi, T. Sakiyama, Y. Kumada, K. Imamura, and H. Imanaka, Curr. Proteomics 5, 161 (2008).
108
K. E. Petersen, Proc. IEEE 70, 420 (1982).
109
S. C. Terry, J. H. Jerman, and J. B. Angell, IEEE Trans. Electron Devices 26, 1880 (1979).
110
M. Li, Sci. China, Ser. G 55, 2316 (2012).
111
Z. H. Wang, Y. H. Meng, P. Q. Ying, C. Qi, and G. Jin, Electrophoresis 27, 4078 (2006).
112
L. M. Lee, R. L. Heimark, R. Guzman, J. C. Baygents, and Y. Zohar, Lab Chip 6, 1080 (2006).
113
J. A. Lee, S. Hwang, J. Kwak, S. I. Park, S. S. Lee, and K.-C. Lee, Sens. Actuators B 129, 372 (2008).
114
A. Iles, A. Oki, and N. Pamme, Microfluid. Nanofluid. 3, 119 (2007).
115
D. J. Harrison, A. Manz, Z. Fan, H. L udi, and H. M. Widmer, Anal. Chem. 64, 1926 (1992).
116
L. Bousse, S. Mouradian, A. Minalla, H. Yee, K. Williams, and R. Dubrow, Anal. Chem. 73, 1207 (2001).
117
N. R. Glass, R. Tjeung, P. Chan, L. Y. Yeo, and J. R. Friend, Biomicrofluidics 5, 036501 (2011).
118
T. Thorsen, S. J. Maerkl, and S. R. Quake, Science 298, 580 (2002).
119
Y. Wang, H.-H. Lai, M. Bachman, C. E. Sims, G. Li, and N. L. Allbritton, Anal. Chem. 77, 7539 (2005).
120
G. Sui, J. Wang, C.-C. Lee, W. Lu, S. P. Lee, J. V. Leyton, A. M. Wu, and H.-R. Tseng, Anal. Chem. 78, 5543 (2006).
121
C. D. Chin, V. Linder, and S. K. Sia, Lab Chip 7, 41 (2007).
122
C. D. Chin, V. Linder, and S. K. Sia, Lab Chip 12, 2118 (2012).
123
C. Kagebayashi, I. Yamaguchi, A. Akinaga, H. Kitano, K. Yokoyama, M. Satomura, T. Kurosawa, M. Watanabe, T.
Kawabata, and W. Chang, Anal. Biochem. 388, 306 (2009).
124
H. He, Y. Yuan, W. Wang, N.-R. Chiou, A. J. Epstein, and L. J. Lee, Biomicrofluidics 3, 022401 (2009).
125
G. T. Hermansson, Bioconjugate Techniques, 2nd ed. (Academic Press, San Diego, 2008).
126
C. Wang, R. Oleschuk, F. Ouchen, J. Li, P. Thibault, and D. J. Harrison, Rapid Commun. Mass Spectrom. 14, 1377 (2000).
127
J. Page, R. Derango, and A. Huang, Colloids Surf., A 132, 193 (1998).
128
J. Guisan, Enzyme Microb. Technol. 10, 375 (1988).
129
J. L. Drury and D. J. Mooney, Biomaterials 24, 4337 (2003).
130
J. J. Park, X. Luo, H. Yi, T. M. Valentine, G. F. Payne, W. E. Bentley, R. Ghodssi, and G. W. Rubloff, Lab Chip 6, 1315 (2006).
131
A. E. Herr and A. K. Singh, Anal. Chem. 76, 4727 (2004).
132
C. Hou and A. E. Herr, Analyst 138, 158 (2013).
133
D. J. Beebe, J. S. Moore, Q. Yu, R. H. Liu, M. L. Kraft, B.-H. Jo, and C. Devadoss, Proc. Natl. Acad. Sci. U.S.A. 97,
13488 (2000).
134
Y. Yang, S.-W. Nam, N. Y. Lee, Y. S. Kim, and S. Park, Ultramicroscopy 108, 1384 (2008).
135
G. J. Sommer and A. V. Hatch, Electrophoresis 30, 742 (2009).
136
Y. Liu, D. Yang, T. Yu, and X. Jiang, Electrophoresis 30, 3269 (2009).
137
M. Harvey, Optimization of Nitrocellulose Membrane-Based Immunoassays (Schleicher & Schuell, Keene, 1991).
138
X. Li, D. R. Ballerini, and W. Shen, Biomicrofluidics 6, 011301 (2012).
139
A. Jane, R. Dronov, A. Hodges, and N. H. Voelcker, Trends Biotechnol. 27, 230 (2009).
140
J. N. Israelachvili, Intermolecular and Surface Forces, 3rd ed. (Academic Press, Waltham, 2010).
141
G. Schreiber and A. R. Fersht, Nat. Struct. Mol. Biol. 3, 427 (1996).
142
P. Gong and R. Levicky, Proc. Natl. Acad. Sci. U.S.A. 105, 5301 (2008).
143
B. S. Jacobson and D. Branton, Science 195, 302 (1977).
144
J. M. Gershoni and G. E. Palade, Anal. Biochem. 124, 396 (1982).
145
H. X. Zhou, Biopolymers 59, 427 (2001).
146
J. G. Franchina, W. M. Lackowski, D. L. Dermody, R. M. Crooks, D. E. Bergbreiter, K. Sirkar, R. J. Russell, and M. V.
Pishko, Anal. Chem. 71, 3133 (1999).
147
C. M. Roth and A. M. Lenhoff, Langmuir 9, 962 (1993).
041501-47 D. Kim and A. E. Herr Biomicrofluidics 7, 041501 (2013)
148
Q. Lan, A. S. Bassi, J. X. J. Zhu, and A. Margaritis, Chem. Eng. J. 81, 179 (2001).
149
K. Tsougeni, P. S. Petrou, D. P. Papageorgiou, S. E. Kakabakos, A. Tserepi, and E. Gogolides, Sens. Actuators B 161,
216 (2012).
150
S. K. Sia, V. Linder, B. A. Parviz, A. Siegel, and G. M. Whitesides, Angew. Chem., Int. Ed. 43, 498 (2004).
151
R. Wang, Y.-L. Yang, M. Qin, L.-K. Wang, L. Yu, B. Shao, M.-Q. Qiao, C. Wang, and X.-Z. Feng, Chem. Mater. 19,
3227 (2007).
152
M. Qin, L.-K. Wang, X.-Z. Feng, Y.-L. Yang, R. Wang, C. Wang, L. Yu, B. Shao, and M.-Q. Qiao, Langmuir 23, 4465 (2007).
153
D. T. Chiu, N. L. Jeon, S. Huang, R. S. Kane, C. J. Wargo, I. S. Choi, D. E. Ingber, and G. M. Whitesides, Proc. Natl.
Acad. Sci. U.S.A. 97, 2408 (2000).
154
A. Bernard, E. Delamarche, H. Schmid, B. Michel, H. R. Bosshard, and H. Biebuyck, Langmuir 14, 2225 (1998).
155
E. Delamarche, A. Bernard, H. Schmid, A. Bietsch, B. Michel, and H. Biebuyck, J. Am. Chem. Soc. 120, 500 (1998).
156
E. Delamarche, A. Bernard, H. Schmid, B. Michel, and H. Biebuyck, Science 276, 779 (1997).
157
A. Bernard, B. Michel, and E. Delamarche, Anal. Chem. 73, 8 (2001).
158
J. Foley, H. Schmid, R. Stutz, and E. Delamarche, Langmuir 21, 11296 (2005).
159
M. Starita-Geribaldi and P. Sudaka, Bioseparation 1, 111 (1990).
160
Y. Lu, W. Shi, J. Qin, and B. Lin, Anal. Chem. 82, 329 (2010).
161
E. Handman and H. Jarvis, J. Immunol. Methods 83, 113 (1985).
162
E. C. Peters, F. Svec, and J. Frechet, Adv. Mater. 11, 1169 (1999).
163
R. B. Bhatia, C. J. Brinker, A. K. Gupta, and A. K. Singh, Chem. Mater. 12, 2434 (2000).
164
C. L. Smith, J. S. Milea, and G. H. Nguyen, in Immobilisation of DNA on Chips II (Springer, 2005), p. 63.
165
S. Knecht, D. Ricklin, A. N. Eberle, and B. Ernst, J. Mol. Recogn. 22, 270 (2009).
166
R. Wacker and C. M. Niemeyer, ChemBioChem 5, 453 (2004).
167
G. Ramsay, Nat. Biotechnol. 16, 40 (1998).
168
A. Larsson, A. Karlsson-Parra, and J. Sj oquist, Clin. Chem. 37, 411 (1991).
169
D. H. Bunka and P. G. Stockley, Nat. Rev. Microbiol. 4, 588 (2006).
170
Y. Xu, X. Yang, and E. Wang, Anal. Chim. Acta 683, 12 (2010).
171
R. K. Mosing and M. T. Bowser, J. Sep. Sci. 30, 1420 (2007).
172
B. Thierry, M. Jasieniak, L. C. de Smet, K. Vasilev, and H. J. Griesser, Langmuir 24, 10187 (2008).
173
G. MacBeath and S. L. Schreiber, Science 289, 1760 (2000).
174
L. Xiong and F. E. Regnier, J. Chromatogr. A 924, 165 (2001).
175
L. Yu, C. M. Li, Q. Zhou, and J. H. Luong, Bioconjugate Chem. 18, 281 (2007).
176
D. Belder, A. Deege, H. Husmann, F. Kohler, and M. Ludwig, Electrophoresis 22, 3813 (2001).
177
T. F. Didar, A. M. Foudeh, and M. Tabrizian, Anal. Chem. 84, 1012 (2012).
178
M. Hu, J. Yan, Y. He, H. Lu, L. Weng, S. Song, C. Fan, and L. Wang, ACS Nano 4, 488 (2010).
179
Y. He, H.-T. Lu, L.-M. Sai, W.-Y. Lai, Q.-L. Fan, L.-H. Wang, and W. Huang, J. Phys. Chem. B 110, 13370 (2006).
180
X. Mao, Y. Luo, Z. Dai, K. Wang, Y. Du, and B. Lin, Anal. Chem. 76, 6941 (2004).
181
B. Thierry, M. Kurkuri, J. Y. Shi, L. E. M. P. Lwin, and D. Palms, Biomicrofluidics 4, 032205 (2010).
182
S. Xie, F. Svec, and J. M. Frechet, Biotechnol. Bioeng. 62, 30 (1999).
183
C. Ericson, J.-L. Liao, K. i. Nakazato, and S. Hjerten, J. Chromatogr. A 767, 33 (1997).
184
T. Konno, J. Watanabe, and K. Ishihara, Biomacromolecules 5, 342 (2004).
185
T. Chen, H. D. Embree, L. Q. Wu, and G. F. Payne, Biopolymers 64, 292 (2002).
186
X. Luo, A. T. Lewandowski, H. Yi, G. F. Payne, R. Ghodssi, W. E. Bentley, and G. W. Rubloff, Lab Chip 8, 420 (2008).
187
C. M. Niemeyer, T. Sano, C. L. Smith, and C. R. Cantor, Nucleic Acids Res. 22, 5530 (1994).
188
L. K. Fiddes, H. K. C. Chan, B. Lau, E. Kumacheva, and A. R. Wheeler, Biomaterials 31, 315 (2010).
189
E. Eteshola and D. Leckband, Sens. Actuators B 72, 129 (2001).
190
N. Stults, P. Lin, M. Hardy, Y. Lee, Y. Uchida, Y. Tsukada, and T. Sugimori, Anal. Biochem. 135, 392 (1983).
191
V. Linder, E. Verpoorte, W. Thormann, N. F. de Rooij, and H. Sigrist, Anal. Chem. 73, 4181 (2001).
192
A. Mantymaa, J. Halme, L. Valimaa, and P. Kallio, Biomicrofluidics 5, 046504 (2011).
193
J. Ylikotila, L. Valimaa, H. Takalo, and K. Pettersson, Colloids Surf., B 70, 271 (2009).
194
A. Dodge, K. Fluri, E. Verpoorte, and N. F. de Rooij, Anal. Chem. 73, 3400 (2001).
195
H. Kaji, M. Hashimoto, and M. Nishizawa, Anal. Chem. 78, 5469 (2006).
196
M. A. Holden and P. S. Cremer, J. Am. Chem. Soc. 125, 8074 (2003).
197
M. Hashimoto, H. Kaji, and M. Nishizawa, Biosens. Bioelectron. 24, 2892 (2009).
198
N. Malmstadt, P. Yager, A. S. Hoffman, and P. S. Stayton, Anal. Chem. 75, 2943 (2003).
199
A. J. Qavi, A. L. Washburn, J.-Y. Byeon, and R. C. Bailey, Anal. Bioanal. Chem. 394, 121 (2009).
200
G. MacBeath, Nat. Genet. 32, 526 (2002).
201
M. A. Holden, S.-Y. Jung, and P. S. Cremer, Anal. Chem. 76, 1838 (2004).
202
C. Priest, Biomicrofluidics 4, 032206 (2010).
203
R. A. ONeill, A. Bhamidipati, X. Bi, D. Deb-Basu, L. Cahill, J. Ferrante, E. Gentalen, M. Glazer, J. Gossett, K. Hacker, C.
Kirby, J. Knittle, R. Loder, C. Mastroieni, M. MacLaren, T. Mills, U. Nguyen, N. Parker, A. Rice, D. Roach, D. Suich, D.
Voehringer, K. Voss, J. Yang, T. Yang, and P. B. Vander Horn, Proc. Natl. Acad. Sci. U.S.A. 103, 16153 (2006).
204
D. L. Huber, R. P. Manginell, M. A. Samara, B.-I. Kim, and B. C. Bunker, Science 301, 352 (2003).
205
S. F. Kingsmore, Nat. Rev. Drug Discovery 5, 310 (2006).
206
S. I. Stoeva, J.-S. Lee, J. E. Smith, S. T. Rosen, and C. A. Mirkin, J. Am. Chem. Soc. 128, 8378 (2006).
207
C. Boozer, J. Ladd, S. Chen, and S. Jiang, Anal. Chem. 78, 1515 (2006).
208
F. Birger Anspach, J. Chromatogr. A 672, 35 (1994).
209
S. Akg ol and A. Denizli, J. Mol. Catal. B: Enzyme 28, 7 (2004).
210
D. Sung, D. H. Shin, and S. Jon, Biosens. Bioelectron. 26, 3967 (2011).
211
D. Sung, S. Park, and S. Jon, Langmuir 25, 11289 (2009).
212
K. Sakai-Kato, M. Kato, K. Ishihara, and T. Toyooka, Lab Chip 4, 4 (2004).